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  • Tierversuch - Donath 1995

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    FeliciaFala-Neguleanu
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    histology for dentistry

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    histology for dentistry

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    © All Rights Reserved
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    47 Ansichten16 Seiten

    Tierversuch - Donath 1995

    Hochgeladen von

    FeliciaFala-Neguleanu
    histology for dentistry

    Copyright:

    © All Rights Reserved
    Als PDF herunterladen oder online auf Scribd lesen
    Markieren Sie unangemessene Inhalte
    von 16
    Preparation of Histologic Sections by the Cutting-Grinding Technique __ for Hard Tissue and other Material not suitable to be sectioned by routine methods — Equipment and Methodical Performance — K. Donath * EXAKT-Kulzer—Publication, Norderstedt 1995 * Professor Dr. Dr. Dr. Kael Donath, Institute for Pethologis, The University of Harbarg CUTTING-GRINDING TECHNIQUE. The cutting-grinding technige in a method used to obtain thin sections (below 10 ym) for" histological examination of specimens which cannot be cut. by con- ventional fochniques (Pig 5). These jnclude such tissues as jaw bones with teeth containing fillings, crowns and bridges, thick cortical bones, implants (motale er ceramics) jaw or long bones or even brittle hypermineralized bone parts. One can see from the proceeding list of specimens shat the cutting gtinding technique is not & substitute for the conventional Cryo-paratfin and hard cutting fechniqnes because mast o° these fissues cannot be ‘imipulntod by the paraffin technique after decaleiti- cation. The solution for this problem has been aporo- ached in different yaya. GROB and STRUNZ (1977) presented their findings using surface siaining on "saw cuts” 30-200 jm thick. Ochers followed these sw cuts with mechanical or marl erincing to reduce the thickness to 30 um (HENSCHKE and FESCH, 1980 etal. The method to be described kere (DONATH and BREUNER, 1982) is roulincly used in daily histolo~ sical diagnosis for, specimens which cannot be pro- sessed in paraffr. Numerons innovations eve been developed through tho routine use of this meted. ‘These include no: only new equipment but new stzi- ning methods and the use of a plastic resin for em- ‘bedding the specimen, To prepare thin slices for histo- logic diagnosis vy ‘ransillumination the folowing machines, aocessories and supplies are needed. Machines LA Bxakt-cutting-grinding system 2.2. Bxalt-micro-grinding eystem Accessories 2a, Eauiht-vacumn-adhesive system device for preparing parallel-sided blocks 2.2 Bxalt-precision-adhesive press for mounting the plexiglass sides 2.5 Baaktigh polymerization unt 2.4 Rrakt-dlehydration- and inditration-umit 25 Brokt-block-drying, ~infltzation, polymerization 2.6 Micrometer serow with digital display 2.7 Fealor gauge 0,05 te 0,59 sam. 28 Straight edge 1. Kulzer Balt: Embedding medium (Technovit 7.200 VLC} Technovit 4000 Technordt light hardening asesive Fixation adhesive (Technovit 7230 VLC) Embecidingemolds (transparent) Flexi slides Abrasive paper (different exits) 2. Others: Glucolmettaerylate Alcohol ‘Mounting media es used in bistology Cover slips by Teehnovit 7.200 VLC Benzine cleaning solution DRSCRIPTION OF THE EQUIPMENT 1.1 Exabt-cutting-grinding eystem (Fig. 2}: The Pukt-oudling-grinding system consists of the out. Ling ust whick is based on the principle of a bandsaw, the precisionparatleling guide with an attachment 10 hold the specimen to bs ent and the cooting wad fu shing system, The speed of the cutting onit can be ad= jusled according to the Tardness of the material in tides to anid the gonerelion of the heat. “The culting bands are staintess-stoc! bands, with a thickness of OL or 02 mm, The entting edge of the saiwblade is imprep~ asiod with diamond or bormitrid partistes. Particle sizes of D 30, D 46, D S4.and D 91 arc evuileble, The cutting wasle doponds on fhe steel-baad aad tie grit size; with a patticle size of 64 approximately 0.250 am is Tort, The precision paralleling guide is ‘eased on the principle of a carriage which ix moved behind the sawbiade by an adjustable weight. In the center of the pacalleling guide is an adjusting screw system vehich can attach a support plate with a mecha- ‘sical clamp or a vacuum plate, 1¢ Lhe serow vlarap ail ‘2 microssrow i atzanged, which provides the object being moved either toward or away from the sawband ‘with 0.02 ram feromenis. Phe cooling-lushinas system consinis of two wiler jets, ene of which is positionod above the objeet to be cut, the other oae below in er- der ta clown the sawbslade. Tae water el for the eaoting system can be regulated, Mechanical Operation “The stmples are placed on the cutting table aud held there by a mechanical clamp, The thle ts wovet tor word the sawblade by weight in a way lal the spect. snc is cul by tho dinnond blade in & veitcal plane. Parallel sives of selectec thickness can be obieined fram the teare specimen. by advencing the mecimen table fn n porponiculnr drcuaon Io the outing plane, fellowing cach eut. Tae debris tha: nesumies ia the chip space hetween the dnnond bits, ring fhe name ing procedure are moved info the Hushing. wystem wherein thshod away via jet of water, Phe dimond ‘bis eventually become clogned because of debris which developed during the process of uxting the hard plastic materia. Theretore, « rough or irregular surlace occurs instoad of a smooth onc, The cutting- nding technique requires a plastis material of ex sremely hard nature, The plastic used for the haré-cut technique is elastic and therclore cyontually clogs ap tho chip space on tho dinmond plato, which ctuses au tineven Surites on the cut thes ad A. Eee Fig t Vewnfrar of Latino shoving the nek cin pntng cen fight stein the pce) and 2 anchner of tke Boeke grading stom SCRIPTION OF THE EQUIPMENT. 1.2 Lxakt-micro-grinding system (1%. 1. The apparstus consists of a main anit with a yotsting grinding plate whioh ia either dimond cuuted end/or covered with sandpaper, The velocity of the grinding plate cam be yegulsted. On top of the main unit, 4 ‘block 4s attached patullel to the grinding plate, The stock moves linearly stimuicted by a winding spindle. _A blanic slide or specimen to be ground ean be atiached te the black by vacuum. The lack alko serves as singe ahd cooling sysiom. ‘The preswure in which the lock wil come in contact with the grinding plate is dotonmined by individual weights, The linene velooty can be regulsted. A micrameter serew is attached ot the blook which menipedlates the thickness of the spect sen fo be gromnd, The misrossrew ii more of less the end point because, os S000 as the determined thickness is reached the automatic grinding mecha aisen ie lopped. Mechanical Operation The thicknest of the object to be ground is mogdlatedt dy adjusting the miciometer screw system attached fo the microsystom, Firs, however, the zex0 point of Fig 2: Ponztnd dae of Recktningrinhng vt wil tebe tase holy (inet and méarmstanea gat on feet pubding devise ench specific grinding specimen needs to be doter- mined using the following described procedure, Zero Point Adjustment (Fig. 2) The appropriate sandpaper and spesimen are plaowd tom the mtero-yrinding syst. White tbe motors are running (gtindiag and perailel system} she microrecter torew is moved towaais the contacts Gndertesth the ‘yt ul they ate barely touching, This coaching point will cause tho molors tu stop and ix considered the determiaed zero point for the chosen (individual) specinien ty register the dhickness to be achieved, Tae ricronneterserew i then tnoved in the opposite dicec- liom unt the sppropslale mumbers appeer on the ine dinar, Now, the misrorstacing system is rod. AS soon as the specimen reaches the programmed thick- how the grinding procaw ix wlopped ‘utoroatcaly, Th has bein proven by experience tbat i boter to ‘use sandpapers of different grits to grind the spovimen aradually, Buerytine, however, the endpaper fs chai fl, the progrumnaing of the anoint and te tice ‘ness adjustmon! af the specimen is mandatory. ‘ie behgroed Coton ts uoed w monn a lactic peeianes hak ert ed DESCRIPTION OF THF FQUIPMENT 21 Usakt-vaceurmadhesive system to mount the specimen lock paratla to the slide (Fig. 32° The adhesive system has 2 lower and upper plate, Both ace conaested by an axle rncunted an ths bottom plate which comtrots the paratfel movement. A vacuum is attached underoath the upper coverplete to held the sido, Operation Procedure The block is to be placed on the lower plate in such a. way that the wide to be cxumined fs directed «ward the lower plate, Underneath the upper plate a slide ‘will be attached with the previous mentioned proce~ dure, the Technovil 4000 used to mount fhe block an lo the slids, is distibuice evenly. At the eed oF the pefymorisation stage, the vacuum will be relcased. Now the block and slide are connected in = parallel Fashion; the slide i ready to be removed ram the upper plale. ‘The specimen, prepared as described above, is 10 be placed to the vacuum appurstes of the Bxakbenicro-grinding systers. The surface of the area of interost witl be ground until paralleling is ‘achieved aad the specimen is at the face af the block. 2.2. Exakt-precision-adhesive press (Hig. 4) ‘Tho mechenical system is similar to the one desotibed in 2, The upper plate however is transparent, (Fig, 4) ‘The "final slide” is fo bo attnohed fo the transparent plate by vaccum. The plate also allows sasio: coattol oe bubble fee gising, Over the upper plate is a light scuree which serves the purpose of "Photopalymeri« zation’. Operation Procedure ‘The pelle slide (final slide) with known thickness iv lightened to the upper plate hy vaerum, The slide with the parallel block ix te be phteed on the mover alle base piste, ‘'hy thickness is 19 be recorded. ‘Fhe Side of interes: will be coated with precision phato= luv. The base plate with the specimen will be prenscd weah uniform pressure by using weights against the upper plate, to iasire hubble free monating the glve should be distributed uniformly within 2-3 eninutes, The potimenzaliom process is inifiaicd by a fight source above th upper plate, The polymerization provess is completed within 810 minutes 24 Micrometer with digital display Precision measurements are essential to obtain scot site resulls, A micromoter with digital display, Mile ‘toy0 2099 ceos digimatie micrometer is provided for this purpose ‘Thee epecifio measuizements sfc required to determine ‘the Kinal thickaess of the specimen, thin section. 1. thickness of specimen block + side = A 2. thickness ef parallel aide = 3 3. thickness of total afer gluing A+ 11+ X= C Thickness of pus (X} will be determined by subtrec- ting A and 3 from C. X=C+ 48) (Ationdion! All cements 10 be measured vhould have the same loraporature ~ de fo hest expansion}. 25 Heeler gauge from 0,95 lo 0,50 eam, ‘Chotrots waste daring separation procedure, Fig, 4: tcahegrcitn adveatve sven for igh tare bowtag ee eect ste Fi, 5: fos sgments inching teeth, prepared Iyaifrett netods fr hsb examination. 1} declticaton of tefvesepinen wi lass ofthe devia enamel, eclodine nbediine and Hemetocsin Eosinstain 1D pasicenbeon win areretment mepared ay amis the thin secon stom, Detcining the dental exam! and calle, idiebive sta, Fig: Wot-bearng lover jaw sepment ofthe mixed Fig, 1: Toth bering epee jaw egnent of 2 men thickness, enitin. 2 um hich ith opening af yy chambers and (Careful eth vith the eatingegrinaing ston, raving te the Bone marr spses fo obiat ter fain, dely= ‘antagan ithe rst moter end muceus manitrane of he aerun, sion pace fran. Fig % Slideof 2 mr thicker af an Fig 10: Crom section thowgh the stank veuilry seg oot cal of hip ondopatheis ceerte i he ers pnt in he right ental fim DESCRIPTION OF THE BQUIPMENT 2.6 Straight edge ‘The siraignt edge is to be used 10 detect uneveness of larger areas; i.e. to eliminate facets or concavities ‘which oscar, especialy on transition areas of metal to bone or to soft tissues, while gringing the specimen with sandpaper. 3. Preparation of the tissues from the fixation Tissue section of 2 to 4 mam thickness can ba fixed ‘well usually wilh good preservation. It is necessary t© cut the tissue im smaller slices, when a histological ‘examination of the pulp of = tooth or the bone marrow Of the lower jaw is requested. (Fy. 7—1.Tissue samp- les from, animal experiments present 10 difficultios, ‘because a perfusion fixetion can be performed. Any tissue which can only be treated by immersion fixation may not be thicker than 2-4 mm. When pre- paring bones, which are surrounded with soft tisse, a prefixation with neutral formalin for 15 to 30 min, at 4? Cis necessary. After pretreattnent with formalin, tho hard and soft tisenes can be sliced in to 2 to 4 mm thick parallel slices without ary problems by using the Exakt-cutting-gtinding system. The maximum size of 2 specimen io 100 x 59 mm. The jaw segmonts exn be held in either a longitudinal or traasverse direction, It is important that the jaw be placed finaly against tho moveable cutting table. The cutting table ia moved toward the sawtand by variable weights. Using this provess. it is possible to cut a jew with teeth and sur- sounding cof tissue into small slices (2 mmm thil). After a fisation periad of 6 to 12 brs, the parallel ‘thin tissue specimen can be further processed by de- clefication or the cutting grinding technique, 4 Dehydration ‘The debycration can be accomplished by either using increasing alcohol or glycolmethacrylate conventza- tions. The time of dehydration in the individual con- centration depends en the thickness of tissue parts ‘With the manual dehydration procedure, 24-32 brs. ate recommended for each step, The dehydration fime can be shortened by using an automatic deky= ration system with agitation and vacuum, 5. Plastic infiltration First step of the plastic infitration is to use a mixture of pure glycolmefhacryiaie and an embedding medium in a rafo LL. The time of infiltration ie about 8 lus ‘when using 2-3 man thick tsswe specimen, The Jength of fime from dehydration to final errbedding can be shoztened to 12 hve, when using ax, autoxiatic eer Deddiag apparatus with agitation anda vacuum syste. 6. Embedding and Polymerization The plastic inflated tissue slices are placed in em- bedding molds. A. 90 called plastic fxation medinm can be used 10 prevent the sa tissue from iting away from the bottam plate. The final polymerization process is accomplished by a light with 450 mim wave Jength, The polymerization takes place in two or three steps. Stop 1: Using low intomity ight an maintaining tempo- ratutes below 40 dogrees Ceatigrace, the embedding media is extensively polymerized, Approzimate time for thie atep ie 4 hours, Step 2: Using a higher intensity blue jight embedding ‘media which has been infitrated into tisme is com- pletsly polymerized. Fer specimens § mm thick or leas ‘the approximate time for this step 1s 4 hours. For spesimen S—L6xmm in thickness the polimerization time should be extended up to 10 hours depending on ‘the actual thickess of the specimen and the type of tissue. Sicp 3: Supplemeatal polymerization using Benzoyl Peroxide In the event thicker specimens (those thicker ‘than 5 mma) contain predominantly soft tissue or high concentrations of blood polymerization ean be ene hhanced by adding Beuzoy! Peroxide to the Kulzer ‘Technovit 7.200 VLC éusing the final infiltration step. After proseeding with the Jirst two steps of paly- merization with the Exaktlght-polymerization unit the specimen can be placed in an incubator et 50 degrees Centigrade for up to 12 hours to complete polymerization. * Wf Benzoyl Peroxide is used, the special safety rules of the county are t be atended. Bet = ae. 19g, 1: Thi econ of bower ow of32yeur- o. Concronents and aque wants tee mermasinal spaces, (dsl the if sent enecope poo Sig subeptbldtftannaaten lyre. thin ween, Dlune be sta a 12. NE ian vite deep packer ‘Thin ection. vane Sa ota) Fig. 25: Hydvoxpapette ererte in dire rrr wi os Cin ection, Blidine [rsenarma Wee”) cones zone (Thia set tine Westin) Pip 20: rt of ig 19, livet conte of craic aed bone: (Thi cet. bie bbe sein) nitration. Ui cts Marr oltre Fig 27: Helenolapae corte fret sme ofthe hone et te gt (Tha sfonde ble iin) PREPARATION TECHNIQUE 7. Preparation of the block to obtain a parallel surface ‘The polymerized tissue blockis removed from tha em- ‘bodiag mold. The nest stop is the preliminary erin ding which brings the area to be examined closer to the surface, In order to obtain a flat surface, the oppo- site side of the plastic block is mounted on a abide by using Technovit_ 4000. Tecinovit 4909 causes no damage fo the slide, The vacumm-adhesive apparatus is used for the mourting procedure, The slide is bold bby vacuum im the upper part of the press. Auto-poly- smerizing resin is piaced an the back side of the speci- men block. Thea, the side of the speaimen to be ax- amined is placed azeiust the lower plate of the appa- rehus, Next the upper plate with the attached sf is towered carefally, untl an adequate contact occurs, ‘the plate then will be secured with a screw. As soon as polymerization is finished the block js ready :o be ‘polished with the micro-grinding tystor. & Preparation of the surface of interest “The block which is mounted on the side is placed iato the yaorum apparatas aifached to the micro paral grinding systean. The grinding table is covered with 1200 grit sandpaper. The grinding process is comple- ted when all the tasue segments io be studied are ex- ‘posed fo the aurface. Simultaneously, the surface of the biock must be paraliel with ibe slide. Unsveness within the surface of the specimen can be dected with 1 etraight edge. Difforences af 3 to 5 ym are accept- able, when large specimens of about 80-100 mm di meter are used, Afler the definitive surface of the specimen to ke studied is reached # is fished with 4000 grit sandpaper to creale es smooth susface as possible, 2-4 reference points must to recorded, 9, Affization of a parallel stide onte the block. ‘The parallelism of the slide must be verified. If dis- scopancies are present, the slide aceds to bo adjusted with the Exakt-micro-grinding system. When paral Jelism is obiained, treatment with sandpaper of 4000 gtit is used to smooth the surface of the slide, The final thickness of the slide is to be recorded in a pro~ tocol (2 measurements). Before the precision affixing, the suriaco of the block and side is 9 bo cleaned 0 ‘with an argcaio cotution (petroleum ot benzine). Don't ‘use any aceion at al. The cleaned slida is to be placed ‘with the side to be mounted upside dowa into the pre- cision prese. ‘Vaownm io used to hold the slide. The precision achesive fs delivered (Chin) onte the prepar ‘ed block surface. The specimen is put om the lower plate. The lower plate is pressed toward the apper ‘Plate after loosing the safety device. The same pres sure is maintained by a variable weight. If bubbles ‘cecur the slide and block can be separated. After clez- ning the sample and side by benzine the same process can be repeated, When perfect mounting takes place, a ‘uniform distribution of the precision adherive in "glue space” will oscur within 2-5 rainy If the adhesion. is satisfactory, photopelymerization is iniated by apply- {og the curing light. The polymerization is completed ‘within 15 to 20 sin, The excess glue, which flows out from the glue space, remains so% because of the in- ‘Buence of the sir (oxygen) and therefore, ean be reeno- ‘ved easly with a towel. Before te separation cul is started, the elue thickness is to be caloulated as descri= bed in 24, R= C-tA+B) 10. Proparation ofthe separation cut by using the cutting-grinding system: Th is possible to place either slide onto the vacmim appasatis attached to the cutting- grinding systam. Be- cause the photopolymerized adhesive area rameins visible, it is acvisible, however, to select the parallel ide. It is helpful to use a paper sticker (200 ym) om the edge of the site to adiust the choosen thickness. While the sambend is slowly moving, the aide is to be moved toward the parallel guidance, by using the thicrometer screw, until the paper sticker barely touches the blade without being damaged. The thick= x0i0 to be expected is about 100 ym, inchading the ‘thickness of the glue. Advancing weights of 50-100 grams are suicient curing the cutting process. Be- cause the finished sido of the alido is placed away from the vacuum system, the cutting process can ve monitored, Any interruptions or changes of the ‘weights while the cutting provedure is in progross ‘will cause uneveness on the two surfaces that ate Produced, Fe. 28: hon section oa cemeved sank tte fru ofa Mp enaresttess. (Canpure Fig 10, Bhi Dive st) ‘Pk 30: Abciar boii Bo pion (ceteris an ectcoblee) al bare dsiucion By esteeblests Ti set, Hrctmatsnsin Bist sitic Fie 33; Aletar bone with sci of tooth ‘Qed margin (end on te surfs of te lon eer. TH rection, Soro Goldner fe Sea s ‘ig 29: Thin mandibuler cross section wth a arew ava pare ‘a osensnihets (Thin sesion. Tdi Bestia) ig 31 Imedertal raion wit bdo Fig. 22: Sebmundidr glo swith xeton afte The elealas op theta Mustaton ofthe moves wid Choe sbgigivally. Ti setion, ene Thin sectioned sen. Fig. 4: Lover ow wits defective ling Pig 35: deals mended (iraigh prc eosylpatite cen verleppingalectar mura. This setion ‘hin sctin, Kose sin PREPARATION TECHNIQUE/STAINING, 11. Preparation of the final thin section The thickness of the saction ia calculated by subtracting the thickness of the paralle; slide including the thick- ness of the glue, from the thickness of the total cut specimen. For example, if the total thickness of the ‘grinding specimens 1655 1m, the parallel slide 1350 pm, siue thickness 5 um — the separated section is 100 wn thio. The calculated value of 160 ym io considered as {he reference point for the micro-grinding system, Be- fore starting the grinding procedure, the zero point in te be detormined. In order to do thet, the slide with the specimen is placed on the vacuum block. The grin- ding table is covered with sandpaper. The specimen is carefully lowered by using the misrometsr screw until the electric curreat for grinding table and paraliel mo- ‘vemnent is interrupted. A coatyol light will indicate that ‘the zeropcint is established. This procedure is perfor- med while the motors are running. Then the actual amount to be ground away is programmed into the micrometer screw system. The apparatuc will be stop- ped automaticaly after the programmed thickness is removed. Ths last polishing of the surface is accom: plished by using sandpaper with a fino grit. Minor grinding marks are efiminated wilh the coverstip me: dium, after the staining procedure. Metals in connec tion with bones and soft tistues must be ground with a diamond coated plate, because sandpager will not provide an absolutely even surface. This phenomenoa ie ed by the fact that metal pressas into the sandpaper, therefore, the surrounding soft and bone tissue will b¢ ground off faster. 12. Staining of thin sections ‘We are rontinely using for the daily diagnostis histolo- ay coction, Toluidine biue because the staining proce- Gare is quick and simple, The metachromatic stain allows corelusions about bone appositions and bone ssonptions, (Fig. Li-18, 19-23, 25, 27-25). ‘Toluidine blue-staining: agitate in 10% HO, 5 mimites rinse in water wipe dry staining in Toluidine blue solution, 20 minutes ive ia water . Wipe dry carefully Ifthe plexighsutide is blue it can be wiped with 70% aleohcl. Not the specimen! 7. Aitec 5 min, drying oF waiting time over night coveralipping with Technovit 7200 VLC. pe ree 2 8, Polymerization on top of the plastic plate of the EXART Precision-Adhesive-Press. Ingredients: Socium Tetzaborat (Boras) Toluidine blue (Chroma) Pyronin-G (Merck 7517) Application: embedded tissue in plastic, are-treated with 1,0 Preparation of 1 liter staining sclution Sotation At Solution B: 800 mil distiled water 200 gal stifled water BgSodium Jetraborat — 2 g Pyronin G 8g Toluidine bine ‘Mix 15 min. with « Mix 15 mia. with & magnetic stirrer, magnetic siorer. Solution A and B are to be mixed with a magnetic stirrer for 15 min. ‘Then mixture js to be filtered 2 x. Of course other tissue-staining methods are possible, ‘when using Techaovit 9.200 VLC. ‘Hacmaioxylin-Bosin (Fig. 39): 1. clean surface of the slide with acetome-atoohot 1:1 2. stain <0 min. in Hasmaizun (Shandon) 3, riave Lmin, in glacial acetic acid (Lut in. 100 mt cist, water) J. water 10 min, in ruanig tao Water: to dye blue stain $ rain, in Bosha (onin-y-water-soluble, Shandor) rinse with cuaning water differentiate briefly in 80 % alcohol . dip the slide quickly in 96% alcobol . dip quickly once ia xylene . covertlip PAS reaction: 10 min, 0.5% petiodit acid solution singe slide im distied water, 2 jars |. place slide for 30 min. ia Sehilfs reagent. place slide 3 x for 2 min, in wash solution rinse slide for 5 min. in running tap water stain 30 min. in Hazrmalaun (Mayer in inewator 60° ©) dye blue 10 mia. in running tap water 8. dhy slide and coverslip STAINING Praduction of wash solution: 30 mi sodium metabiaulfte (Nay $50) and 30 ml of LN hydroctloric zeid in 600 mnl distilled water Ingredients of the Schiff’s reagent Pararosanilin (Sigma, P 1528, Merch) Hydrochlone acid (IN) Sodium metabisulfite Active charcoal distilled water Production of Schiff reagent Dissolve 10 g Pararosanilin in 2L flask by adding 2000 distilled water cool dawn to 50°C. Ada 209 ml:1 N'HCI and coal down to 25? C. Dissclve ia this sotation, 10 g (Na,5,05) Place for 24 tre ia dark environment. Then add 10 g active charcoal, mix well, ‘After ftration store ina drown bottle, Astra Blue stain (Fig. $2). 1. 3mnin, in 3 % acetic eid 2. stain 30 min. in Astra Blue solution, 3. cinse with tap water 4, stain for 30 min, in Kemechtrot 5. nse with tap water 6. dey slide and coverslip Ingredients: Astra Blue (1278 Mcrok) Kemechtrot (15939 Merci) Production of Solution: 1. Astra Bue solution: dissolve 2 g Astra Blue in 100 mal 3 % avetic avid, overnight. 2, Kemecktrot (musloar fast xed staiz): 1g Kernechtrot dissolve $0 g aluminum sulfate in 1000 mot Aestilled water; filter after cooling down, Masson-Trichrom-Goldner (Fig. 24, 23) stain 1S min. Weigerts ferric Haematoxylin nse 5 main. in ramming tap water stain 7 min. in Masson-sclution (Goldner 1) rinse with 2% acotic acid stain 3 min. phosphormolybdaenunr-acid- Orange-G rinse with, 2% acetic acid stain 15 min. in "igh green’, 60° C incubator rinse with 2% acetic acid . dip slide briefly in distiled water, to wash off the acetic avid 10. dry slide end coverslip ‘To consider: Prepare Weigerts ferric Haematoxylin, shartly before use. Use oqual parts of | and 2 solutions, weppe vane Ingredients: Acid fuchsin (Rubin 8, Merck) Xylidin Ponceau (1 B 207, Chroma) ‘Azophloxin (1B 103, Chroma) Light green (yellowish, Merck) Production of the solutions: A Masson-Solution (Golder D 1. dissolve 1 g acid fochsin in 100 ml distilled water acid 1 al glacial acetic acid 2, cissolve 1 g Kylidin-Foncean in 100 mal distilled. water and 1 ml gacial acetic acid 3, dissolve 0.5 Azophlosin in 10€ ml distilled water and 02 ml glacial aoetic acid. ‘Mix 33 sl of the acid fuchsin in solution and 66 ml Xylidin-Popceau-solution. Mix 100 ml of this solution with 20 ml Azophloxin, solution and 880 mil 0.2 % acetie acid. ‘The finished mixture is identical with me Masson- Goldner-Solution (Goldner). B. Orenge-G J. dissolve 10 g Orange-Gi in 500 mi distilled water 2. dissolve 15 g Motybdlatophosphor acid (Merck) in above (1) solution. C, Light green 1. mix 0.5 g light green and | mil of placial acetic acid into $00 ral distilled water, B STAINING. Silver stain of Gomori: 1. 3 min, 0.25 % potassium permanganate solution 2. rings with distilled water 3, bleech in 3 % potassium metabirulfite-solution 4. water 5 tain, in tap water 5, sensitize Lmin in. 1 Sh fervic ammonia alaun- solution ‘water 2 mia. it tap water rinse with distilled water, 2 jars impregnate 1 min. insilrer solution . dip in distilled water (5-10 see.) 10, reduce 5 mia, in 46 newtral formalin 1. zinge with distiled water, 2 jay each 3 aca 12. efld 5 min. 0.1% anrous chloride 18. rinse with dlstiled water 14. reduce 1 min, in 3 % potassium meta-bisalfite- solution 15. sinse with distilled water 16. fix 2. mia. in 5 % sodiumtiiosulfate-solution 17. water 5 tin. in tap water 18, stain 40 min. Kemechtrot 19, rine with distifed water 20, dry slide and coverstp Production of silver tolution: 10 ml of a.20 % solution of silver nitrate, a brown pre- Sipitation developes. Ada drop by drop liq. ammon’- caustci, until she precipatation just disappears. Fill up with distilled water to the four fold. Ingrediontst, Kemocktrot (15939 Merek) Gold chloride = sodium tetractlor-aureat (ZI) (60550 Fluka AG Switzerland) Movat (silver impregnation): 20 min. 0.5 % periodic acid . water in distilled water, 2 jars each 5 mia, 5. stain Jox TY? brs. insilver solution, 60° Cin incubator |. rinse with distilled water, 2 jars Sin, 39 sodium thiosulfate 5. water 5 min, tap water dry slide and coverslip Mavaepe is Froduotion Gf silver solution: Add to 40 ml of 2 3 % heramsthylentetramin-solution 5 ml of 2 5% silver nitrate-solution, mix well. Add Sl of 22 % borate solution; after 5 min. filter 2 x Giemsa Fg. 3D 1. stain 15 ¢0 30 min. in giemsa-solutioa, 60°C incubator 2. rinse with distilled water 3. dipa few seconds ma cuvette containing dacial acid waver (8 drops glacial acid in 100 ml distilled water) 4. 3 cuvettes containing 96 % [sopropylalcohol. ‘keop in lst cuvette for 5 min.) 5. 3.x for 2 min, isopropanol 6. dip briefly m xylene / soveralip aide Substrat: CHiemsa-Solution (9204, Merck) ‘Yan Gioson-connective tissue stain (1g. 35) . clean alide with aostone-elookol (ls) , stain [5 mcin, in Welgert’s fertic Haematoxylin water 5 min. in running tap water |. stain 4 min, in van Gieroa-mbeture (pierotvcnein) 5. rinse with water 6. difforontite briefly in £0.% alectaoh, 1, dip once in 96% alcohol 8. 9. Appe dip once in xylene coverslip ‘Noetive: set ap Weigerts ferric Haematoxylin just before to de used (Solution { and 2 equal amount) Production ofthe ven Gieson maintare: 100 ml tered saturated, aqueous peri acid + 10 ml % acid fichsm solution, seat Tashi (1 525, Chroma) STAINING. Combined Blastin-ran Gieson-stain: . clean slide with asetone-alcohol (11) 9. stain 30 min. in Resorcin-Fuchsin, according to Weigert . riage with tap water differentiate briefly in 0.5 % HCL-alcohol vwater 10 mip. in running tap water 5. T min, axdesr staining sosording to Weigerts ferric Haematoxylin 7, water 10 min, in nmning tap water 3 stain 4 min, in van Gioson-misture 9. rinse with tap water 10. differentiate shorty in €0 % alcohol LL. dip once in 96 % alcohol 12, dip once in ayfone, 13, coverslip Reagents: Resorcis- Fuebsin (11430, Chroma) ‘Van Gieson-mintare (see recige van Gissox) Resorcin-Fuchsin alter Weigert: Dissolve 1 g Resorcin-Fuchsin in 4 ml 25% nitrite avid and fil up to 566 rl with 79% alcohol. Azan of Heldenhain: 41, 5 min, Aniline-aleohol ({ ml Aniline-oelin 1000 onl dlisted water) 2, dip briefiy in 80 % aleatol 4. rinse with distilled waver 4. 30 min, muclear staining with exercarmine, incuibator 60° 5. nse with distilled water 6. differeatiats in Aniline-

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