Protocol 1.

Preparation of tissue specimens for light microscopy

Equipment and reagents

 Automated tissue processor (Hacker) or glass beakers

 Water-bath
 10% formalin in physiological saline
 740P IMS (BDH)
 Xylene (BDH)
 Low melting point wax

Method
1. 10% formal saline, at room temperature, for 0.5 h.
2. 70% alcohol, at 40oc, for 1 h.
3. 90% alcohol, at 40oc, for 1 h.
4. 740P IMS, at 40oc, for 0.75 h.
5. 740P IMS, at 40oc, for 0.75 h.
6. 740P IMS, at 40oc, for 0.75 h.
7. Xylene, at 40oc, for 1 h.
8. Xylene, at 40oc, for 0.75 h.
9. Xylene, at 40oc, for 0.75 h.
10. Wax, at 60o c, for 5.5 h.

Protocol 2. Haematoxylin and eosin stain (H&E)

Reagents

Xylene (BDH)

740P IMS (BDH)

Harris’ haematoxylin staina

Method

1. Take sections to water:

(a) Dewax in xylene for 3-5 min.
(b) 740P for 10 sec.
(c) 90% alcohol for 10 sec.
(d) 70% alcohol for 10 sec.
(e) Running tap-water for 10 sec.
2. Stain with Harris’ haematoxylin for 5 min.
3. Wash well with running water.
4. Differentiate with 1 % acid alcohol
5. Wash and blue in running tap-water for 5 min.
6. Stain in 1% eosin for 5 min.
7. Wash in tap-water for 5 min.
8. Dehydrate in graded alcohol:
 (a) 70% alcohol for 10 sec.
(b) 90% alcohol for 10 sec.
(c) 740P, twice, for 10 sec.
9. Clear in at least two changes of xylene.
10.Mount in suitable mounting medium.
a
The stain, as bought from the manufacturer, is a poor dyeing agent; to obtain purposeful
results it needs to be converted to its oxidation product haematein. This is achieved by the use
of an oxidizing agent such as mercuric II oxide (HgO). Before conversion, haematoxylin is
added to an aqueous solution of alum (usually the potassium salt-aluminium potassium
sulfate).This is the mordanting step, where aluminium ions combine with the haematoxylin
dye; the resultant aluminium-haematein complex then stains via the metal ion Al2+.

Protocol 3. Silver stain for nucleolar organizer regions (AgNORs)

(7)
Reagents

 Solution A (50% silver nitrate): 50g AgNO3

(BDH) in 100 ml distilled water
 Solution B (gelatin solution): 2 g gelatin
(BDH), in 1 ml formic acid (BDH), 100 ml
Distilled water
 Working solution: solution A (2 parts): Solu-
tion B (1 part), mix immediately before use

Method

1. Take sections to water:

(a) Dewax in xylene for 3-5 min.
(b) 740P for 10 sec.
(c) 70% alcohol for 10 sec.
(d) 90% alcohol for 10 sec.
(e)Running tap-water for 10 sec.
2. Rinse sections in distilled water for 2 min.
3. Incubate in freshly prepared working solution for 45 min at room
temperature.
4. Wash in distilled water for 1 min
5. Dehydrate in graded alcohol:
(a) 70% alcohol for 10 sec.
(b) 90% alcohol for 10 sec.
(c) 740P twice for 10 sec.
6. Clear at least two changes of xylene.
7. Mount in suitable mounting medium.
8. AgNOR sites are intranuclear black dots. The background is pale
yellow.
 Protocol 4: General immunoperoxidase method

Reagents

 Dako pen grease-type pencil

 Trypsin (Sigma):0.05 gtrypsin, 50 ml 0.1%
Calcium chloride (BDH)
 Tris-buffered saline: 30.285 g Tris, 16.875 g
NaCl (BDH), 187.5 ml 1 M HCl, distilled
water to 5 litred, adjust pH to 7.6, 0.5 ml
Tweena
 TBS/0.1% bovine serum albumin (BSA)/
0.1% sodium azide: 500 ml Tris buffer
0.05 g BSA, 0.05 g sodium azide (BDH)b
 Swine serum: 5 ml normal swine serum,
20 ml Tris buffer pH 7.6
 Diaminobenzidine tetrahydrochloride (DAB):
50 ml Tris buffer, two DAB tabletsc
 Copper sulfate (BDH): 4 g copper sulfate,
7.2 g sodium chloride, distilled water to
1 litre
 Primary and secondary antibody kit (Dako)
 Streptavidin (Dako)
 Horseradish peroxidase (Dako)

Method

1. Take sections to alcohol (see protocol 2, step 8).

2. Ring sections with Dako pen. This is to restrict the antibody staining
to the section only.
3. Take sections to water (see protocol 2, step 1).
4. If trypsin is required, place sections in Tris buffer pH 7.6, for 5 min,
then trypsin in calcium chloride 37oc ,pH 7.8, for the required time
according to the antibody used.
5. Rinse in distilled water.
6. Block endogenous peroxide in 3% hydrogen peroxide for 5 min.
7. Rinse in buffer.
8. Immerse in swine serum for 10 min.
9. Drain off swine serum
10. Incubate with primary antibody diluted in TBS+0.15 bsa AND 0.1%
sodium azide for 30 min at room temperature.
11. Incubate negatives in TBS/BSA/azide for 30 min at room temperature.
12. Rinse briefly in TBS for 1-2 min.
13. Incubate with secondary antibody diluted in TBS for 30 min at
room temperature; poly-biotrinylated goat anti-rabbit 1:500, mono-
biotinylated goat anti-mouse 1:300.
 14. Rinse briefly in TBS for 1-2 min.
15. Poly and mono kit. To 5 ml Tris buffer add one drop streptavidin
and one drop biotinylated horseradish peroxidase.d Incubate for 30
min at room temperature.
16. Rinse briefly in TBS for 1-2 min.
17. To 5 ml DAB in TBS add 20 ml of 3 % hydrogen peroxidee for 10 min at
room temperature.
18. wash in tap-water for 5 min.
19. copper sulfate solution fpor 1 min.
20. wash in tap-water for 5 min.
21. counterstain with Harris’ haematoxylin (see Protocol 2, steps 2-5).
22. Complete mounting procedures as in Protocol 2, steps 8-10.
23. Antigenic sites stain brown. Nuclei stain blue.
a
Do not add Tween if oestrogen antibody receptors are the target antigen.
b
Store in fridge.
c
Store in freezer.
d
Leave to stand for 30 min before use.
e
Filter before use.

Protocol 5. Procedure for obtaining kohler illumination

Equipment

 Light microscope with transmitted ligh source

 Phase telescope
 Slide with good contrast

Method

1. Switch on the light source and centre it if the microscope lamp has
adjusters.
2. Place a slide with a tissue section on the microscope stage and obtain
an image using ax10 objective.
3. Partially close the field iris until it appears in the field of vindary is just ouew. Focus
the image of the iris using the sub-stage condenser height adjustment
control.
4. Centre the image of the field iris using the substage condenser
Centring screws.
5. Open the field iris until its boundary is just outside the field of view.
6. Take out an eyepiece and replace it with a phase telescope focused on
the back focal plane of the objective lens.
7. Close the condenser iris until it is about 70% of the back focal plane of
the objective lens.
8. Replace the eyepiece.
9. When changing objectives the illumination will need to be rechecked.
Protocol 6. Calibration of the light microscope

Equipment

 Light microscope
 Stage micrometer
 Eyepiece graticule

Method

1. Set up the microscope to achieve kohler illumination (Protocol 5).

2. Obtain an image of contrasty specimen using the specimen using the objective lens
required to make measurements.
3. Replace the slide with a stage micrometer and focus onto the scale.
4. Focus the image of the eyepiece graticule by turning the focus ring of
the eyepiece until the image of the graticule is sharp against the image
of the stage graticule.
5. Replace the eyepiece of the microscope and align the zero mark of the
eyepiece graticule with the zero mark on the stage micrometer.
6. Count the number of divisons on the eyepiece graticule between the
zero and the next convenient mark of the stage micrometer.
7. Calculate the distance on the stage micrometer and divide the num-
ber of disvisions on the eyepiece graticule to obtain the dimensions of
One eyepiece graticule division.

Protocol 7. Measurement of mean nuclear volume using the

point intercept method

Equipment

 Overlay graticule of known dimensions

 Stage micrometer
 Suitable eyepiece graticule
 Micrograph of known magnification

Method

1. Superimpose on the micrograph a graticule consisting of a series of

equally spaced parrel lines (see Figure1 ) or, if measuring directly
from the microscope, superimpose the image of the calibrated eye-
piece graticule on the image (Protocol 6). The light microscope must
be aligned to achieve kohler illumination (Protocol 5).
2. Measure the total length of line (intercept inside each nucleus. If more
than one line intersects a nucleus, measure the length of each inter-
 cept and add the two together.
3. Raise each intercept length to the power three.
4. Calculate the mean of the cubed intercept lengths.
5. Multiply the mean value by π/3 to obtain unbiased estimate of mean
nuclear volume.