Beruflich Dokumente
Kultur Dokumente
14/12/16
be located thus, the astrocytes present in the section cannot be identified and their distribution
in the sagittal mouse brain section cannot be observed.
Conclusion:
As predicted from the results of this practical, features of the sagittal mouse brain can be
identified such as the hippocampus, the cerebellum and the corpus callosum. This meant it
was possible to estimate the location of other sections and features of the mouse brain. As
previously mentioned in the result section of this report, the fibrous astrocytes could not be
observed and therefore GFAP could not be located. The protoplasmic astrocytes present in the
section of adult sagittal mouse brain have a lower expression of GFAP and therefore could
not be observed.
Appendix
Materials
Youhavebeenprovidedwithaslidecontainingasagittalsectionofanadult
mousebrain.Itisaparaffinembeddedsectionthathasalreadybeendewaxedand
rehydrated.ItisprovidedinTrisBufferedSaline(TBS)inaCoplinjar.
Solutions
Note:onlypartialinformationisgivenonthecompositionofthesolutions.Itisnot
necessarytoknowdetailsoftheconstituents
Intubes:
1.Redtube:Blockingsolution(2%BovineserumalbumininTBS)
2.Yellowtube:Primaryantibody(RabbitantiGFAPIgG,predilutedto1:500in
TBS)
3.Greentube:Horseradishperoxidase(HRP)conjugatedsecondaryantibody
(SwineantirabbitIgG,conjugatedtoHRPandpredilutedto1:100inTBS)
4.Cleartube:Diaminobenzidine(DAB)solution(DABdilutedinTrisand
includingH2O2asasubstrate)
InCoplinjars:
1.TrisBufferedSaline(TBS)
2.Scottstapwater
3.70%Alcohol
4.90%Alcohol
5.Absolutealcohol(100%ethanol)
6.Histoclear
Other:
1.SmallbottleofGillsHaematoxylin
2.SmallbottleofDPX
Protocol
Pleaseensurethatyoustainthesideoftheslidewiththetissueonit!
1.Placeyourslideontherackontheplastictrayandusingadisposablepipette,
addsufficientBlockingsolution(Redtube)justtocoverthesectionentirely.Leave
for3minutes.
2.Drainoffthesolutionintotheplastictray.Thesectionisinthemiddleofacircle
thathasbeenmarkedoutwithapenthatgeneratesawaterresistantfilm.This
meansthatasolutioncanbeappliedwithinthisringwithoutspreadingouttothe
restoftheslide.Thisisimportantwhenapplyingexpensiveortoxicsolutionsto
keepthevolumesusedtoaminimum.Thususingadisposablepipette,coverentire
section(notentireslide!)withinthewaterresistantringwiththerabbitantiGFAP
antibody(Yellowtube).Leavefor30minutes.
3.DrainoffthesolutionintotheplastictrayandplacetheslideinTBS(inthe
Coplinjarthatyouoriginallytooktheslidefrom)for3minutes.
4.DrainofftheTBSintotheplastictrayandreplacetheslideontherack.Forthis
andallsubsequentstepsusingthesliderack,makesurethesectionisuppermoston
theslide!Usingadisposablepipette,placejustenoughoftheconjugated
secondaryantibody(Greentube)ontheslidetocovertheentiresection(seenote2
aboveaboutapplyingsolutionswithinthewaterresistantring).Leavefor30
minutes.
5.WashagainbyreturningtheslidetotheCoplinjarcontainingTBSfor5
minutes.
6.DrainofftheTBSintotheplastictrayputtheslidebackontherack.Usinga
disposablepipette,covertheentiresectionwiththeDABreagent(Cleartube).
Again,seenote2aboveaboutapplyingsolutionswithinthewaterresistantring.
RememberthatDABisverytoxicandsodothisstepCAREFULLY.Leavefor10
minutes.
7.HoldtheslidewiththeforcepsprovidedanddraintheDABreagentbytapping
theslideonto3or4sheetsofthetissuesprovided.Again,performthisstep
carefullyandensurethatthesmallamountofwasteDABisconfinedtothetissues.
PlacetheslideintothejarcontainingTBSandleavefor1minute.
8.DrainofftheTBSintotheplastictrayandputtheslidebackonthesliderack.
UsingadisposablepipetteaddenoughGillshaematoxylinfortocoverthesection.
Leavefor30seconds.
9.DrainoffthehaematoxylinintotheplastictrayandplaceslideintheCoplinjar
containingtheTBS.Usingtheforceps,lifttheslideupanddownintotheCoplin
jar3timestohelprinseoffthehaematoxylinandthenleaveitintheTBSfor1
minute.
10.DrainofftheTBSintotheplastictrayandplacetheslideintotheCoplinjar
containingScottstapwaterfor2minutes.
11.Withtheslidelabelfacingtheblottingpaper,gentlylowertheslideandpress
thebackoftheslidetoensurethatallwaterisabsorbed.Dothissmoothlyanddo
NOTmovetheslideinanydirectiononceitisincontactwiththeblottingpaperor
youmayloseordamagethesection.Simplygentlylowertheslide,pressonthe
backofitandgentlyliftstraightupagain.
12.Wipethebackoftheslidedrywithatissue,takingcarenottotouchthesection
ontheotherside.PlaceintheCoplinjarcontaining70%alcohol.Leavefor2
minutes.
13.Draintheexcessalcoholintotheplastictrayandthenplacetheslidein90%
alcohol.Leavefor2Minutes.
14.Draintheexcessalcoholintotheplastictrayandthenplacetheslidein
absolutealcohol.Leavefor2Minutes.
15.Holdingtheslidevertically,brieflydrainbyplacingtheendoftheslideaway
fromthelabelontothesurfaceoftheblottingpapersothatonlytheedgeofthe
slideistouchingandtheabsolutealcoholisdrainingdowntheslideontothe
blottingpaper.ThenplacetheslideintheCoplinjarcontainingHistoclearand
leavefor2minutes.Agitateevery30secondsbyraisingandloweringtheslidein
theCoplinjarabout5times.
16.Checkyourslide.Ifthesectiononithasawhiteemulsion/film,thengobackto
stage14andcontinuefromthere.
17.ApplyoneortwosmalldropsofDPXmountingmediatothesection.Transfer
theDPXusingthesmallwoodenstickprovided.TheDPXwillclingtothestick
andthenfalltotheslide.Donottouchthesectionwiththestick.Carefullylaya
coverslipontheslide,coveringthesection.Oncethecoverslipisinplace,donot
moveitasyoumaydamagethesection.
TheDPXonthesectionwillbelefttopolymeriseandyouwillbeviewingyour
ownsectionsinthepractical:Immunohistochemistry:followup.
.
Marked by:
Date marked:
Student Number:
(18-20)
(16-17)
(14-15)
(13)
(12)
(10-11)
(8-9)
(<8)
Poor
B+
Adequate
Good
A+
Excellent
A++
Material chosen
Factual content
Irrelevant
Inaccurate, many errors
Understanding
Logical, well-argued,
supported by evidence
Explanations
Marker
Comments
Illogical, unclear
Difficult to understand,
confused, vague
Poor quality, unclear,
redundant
Informal and lay terms used
Too difficult or too simplistic
Good points: