Appendix

1) Esposti’s fluid

Mix together:

100 ml distilled water
45 ml methanol
10 ml glacial acetic acid
Store at ambient temperature

 

2) Buffers

0.01M phosphate-buffered saline (PBS), pH 7.2 -7.4.

This buffer can be made from purchased tablets (ensure the molarity and pH are correct) or more cheaply made in the laboratory.

Reagents

Sodium chloride, 8.7g
Potassium dihydrogen phosphate, 0.272g
Disodium hydrogen phosphate, 1.136g
or
Disodium hydrogen phosphate.2H2O, 1.41g
or
Disodium hydrogen phosphate.12 H2O, 2.83g

Method

Dissolve the salts separately in distilled or de-ionised water (sodium phosphate dissolves more easily in hot water) then mix, make up to 1 l and check pH.
A 10-times concentrated solution may be made (0.1M) and diluted with water for use. The pH of this should be checked when diluted as the pH of the concentrate will be different.
Store at ambient temperature

 

0.05M Tris-buffered saline (TBS)

Reagents

Tris(hydroxymethyl)methylamine  6.07g
Sodium chloride, 8.7g
Concentrated hydrochloric acid

Method

Dissolve the salts in 900 ml distilled or de-ionised water. Add concentrated HCl until the pH reaches 7.6. Make up to 1 L with water.

 

3) Antibody diluent

PBS (as above) containing 0.1% bovine serum albumin and 0.1% sodium azide.

 

4) Antibody storage

Many antibodies can be kept for long periods in the diluent at 4°C but the solution should be tested at intervals for immunoreactivity.  Normal serum for blocking can also be stored in this solution.

Do not use the diluent  for peroxidase-labelled antibodies as the enzyme is inhibited by sodium azide. Use PBS alone for dilution and do not store diluted antibody.

Most unlabelled  antibodies can be stored frozen at -20°C, but divide the stock into suitable aliquots before freezing to avoid continual freezing and thawing. Rapid freezing and thawing once only is preferred. If possible, snap-freeze the aliquots  in liquid nitrogen before placing in the freezer. Do not freeze antibody solutions at a higher dilution than 1/100.

Some antibodies are provided diluted 1:1 in glycerol. This prevents the antibodies freezing solid and the stock can be taken in and out of the freezer without harm. The dilution must be taken account of when the working solution is prepared.

 

5) Blocking endogenous peroxidase activity

Hydrogen peroxide in methanol/PBS is recommended for cell preparations.

Reagents

30% hydrogen peroxide, 1 ml
70% methanol in PBS, 100 ml
(0.3% hydrogen peroxide)

Method

Make the H2O2 solution just before use. Immerse preparations for 30 minutes then rinse in PBS. 

 

6) Heat-mediated antigen retrieval

0.01M Citrate buffer, pH 6.0

For 2 L:

Reagents

Citric acid, 3.8g
2 M sodium hydroxide (8% aq)

Method

Dissolve the citric acid in 1.9 l water
Add the sodium hydroxide solution (stirring) until the pH reaches 6.0.(20-30 ml will be required). Make up to 2 l with water.
The buffer can be kept at room temperature for several days or stored in a refrigerator at 4°C.

 

Tris/EDTA buffer, pH9.0

Reagents

For 1 L (10 times concentrated)
Tris(hydroxymethyl)methylamine,  12 g
Ethylene-diamine tetra-acetic acid, disodium salt, dehydrate, 1 g
1 M hydrochloric acid, about 500 ml
Water, 500 ml

Method

Dissolve the salts in the water.
Add the hydrochloric acid, stirring and checking the pH to pH 9.0
This buffer solution is 10 times concentrated. Store at 4°C and dilute as required.

 

Microwaving method*

Using a domestic microwave oven with a revolving plate

  1. Place in the microwave oven 400 ml of the buffer in a covered plastic container together with 200 ml of distilled water in a plastic beaker. Heat for 2 mins at 750 W. The solutions should reach 40 – 60°C. Remove the water and keep aside for topping up.
  2. Place in the buffer a plastic rack carrying the preparations , ensuring that the slides are covered. Put a lid on the container or cover loosely with “cling-film”. Mark the liquid level on the container.
  3. Microwave at 750 W for 5 minutes. The solution should boil.
  4. Check the level and top up to the original mark with the warm distilled water if necessary (take care about steam when removing the lid of the container).
  5. Repeat steps 3 and 4 for the required length of time – usually 10 to 30 minutes but this should be assessed for each antigen.**
  6. Using insulated gloves, remove the container to a sink and carefully remove the lid. Run in cold water until the rack of slides can be lifted out without danger of the buffer crystallising on the preparations. Rinse in tap water, then place in PBS.
  7. Rinse in water then transfer to PBS and continue with the immunostaining method.

*There are many ways of organising HIER. This is the one used in the authors’ laboratory.

**If preparations are being immunostained for antigens requiring different heating times in the same run, begin with those requiring the longest time then add the others at the appropriate stages. This avoids having to remove slides from the hot solution resulting in precipitation of the buffer salts on the preparation.

 

7) Diaminobenzidine solution for peroxidase development (Pelliniemi et al, 1965)

Incubating solution:

Reagents

3,3’-Diaminobenzidine tetrahydrochloride (DAB) (e.g. Sigma-Aldrich D5637)  50 mg
PBS 100 ml
Hydrogen peroxide (30%), 30 µl (final concentration 0.03%)

DAB can be purchased as tablets or as solutions to be added to buffer. If you wish to make your own, buy 5g or 25 g quantities and to avoid weighing the powder dissolve the entire amount in distilled water to give a solution containing 50 mg DAB/ml. Stir (covered) for 15 minutes in a fume cupboard, then dispense into vials containing the appropriate volume for your use. For example, 5 ml aliquots to make 500 ml or 1 ml aliquots to make 100 ml of the final 0.05% incubating solution. Cap the vials and place in a -20°C freezer for storage. For use, thaw a vial and add the DAB to the appropriate volume of PBS. Add hydrogen peroxide to give 0.03%, mix well and incubate the slides for 10 minutes.

Rinse in PBS then water, and proceed to the nuclear stain.

Safe disposal of DAB

DAB must be disposed of according to local safety regulations. It is a potential carcinogen and must not be disposed of into the waste water system.

 

8) Immunostaining procedure – manual method

Fixed cell preparation on slide, PEG (if used) removed in 2 changes of 95% methanol). Include positive control slide and negative control of the preparation being tested.

N.B. Steps 1 and 2 may be done in the reverse order if preferred.

TBS may be substituted for PBS throughout if preferred.

  1. Block endogenous peroxidase-like enzymes
    Immerse slides in 0.3% hydrogen peroxide in 70% methanol in PBS (1 ml of 30% H2O2 in 100 ml 70 % methanol in PBS) for 30 minutes. Rinse briefly in PBS.
  2. Antigen retrieval see above
  3. Taking care that the preparation does not dry, dry round area of preparation, draw circle round it with isolating pen, place on a rack in a humid chamber and apply background- blocking serum (normal serum from species providing the secondary antibody, diluted 1/10 in antibody diluent). Cover the incubating chamber and leave for 10 minutes. Drain off the serum and rinse briefly in PBS.
  4. Dry the slide except for the area of the preparation. Drain off as much buffer as possible from the preparation (to avoid diluting the antibody that will be applied next) and ensure that the insulating circle is dry. Replace slide on the rack in the humid chamber and apply optimally diluted primary antibody. The exact quantity is not important but the antibody must cover the area of the preparation. Repeat with the positive control preparation (one for every antibody being used) and with a negative control preparation (one for every case being tested) using PBS instead of the primary antibody. Replace the lid and leave to incubate for one hour at ambient temperature or overnight at 4°C according to the optimised procedure.
  5. Drain off the primary antibody and place the slides in a rack in PBS. Rinse in three changes of PBS for 5 minutes each.
  6. Dry the slides as before and place in the humid chamber (at ambient temperature). Apply the labelled secondary antibody (anti-mouse Ig or anti-rabbit (or other species) Ig at the optimum dilution for 30 minutes or as recommended in the manufacturer’s instructions. Some kits provide a “universal” second antibody that will react with both rabbit and mouse immunoglobulins.
  7. Rinse in 3 changes of PBS, then dry the slides and replace them in the chamber as before. Develop the peroxidase according to the kit instructions (usually 5 to 10 minutes. It is important always to use a uniform development time, in order to compare immunostaining intensity between samples and antigens. N.B. Wear gloves when dealing with DAB – it is a potential carcinogen.
    For “home-made” developing solution see appendix)
  8. Drain off the DAB (For safe disposal of DAB solutions, see appendix)
    Rinse slides in PBS then running water.
  9. Counterstain nuclei lightly with haematoxylin in the usual way, differentiating in acid alcohol and “blueing” if necessary. Dehydrate through graded alcohols, clear in xylene and mount in synthetic resin mountant.

 

9) Semi-automated method using Sequenza™ racks

The steps of the method are the same as for the manual method, except that steps 1 and 2 are probably best done in the reverse order. The slides can be inserted in the Sequenza racks after the antigen retrieval step and the endogenous peroxide blocking done in the racks. The rinsing steps are for 5 minutes each and are done with PBS containing 0.05% Tween 20. This is a detergent which helps the buffer to run smoothly down the slide. The space at the top between the slide and coverplate is filled with the buffer (from a wash-bottle) and will take 3 to 5 minutes to run through. Ensure visually that all the buffer has run through before applying the antibody solution to avoid diluting it.

Each antibody step will require 100 µl of diluted antibody per slide.

The peroxidase development and nuclear stain (use Mayer’s Haemalum, which does not need differentiation) can be done on the Sequenza racks, once the appropriate time of incubation has been standardised. Dispose of the waste solutions in the racks in the same way as the used DAB solution from the manual method.

After this stage, remove the slide/coverplate combination from the rack, separate the two under the surface of water in a dish, place in a slide rack and dehydrate, clear and mount as usual.

If the coverplates become discoloured with DAB, place them in water with added bleach (sodium hypochlorite) overnight. Take care not to let the smooth surface of the coverplates become scratched. Rinse them in distilled or deionised water and place them individually to dry before replacing them in their storage box.

 

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