100 ml distilled water
45 ml methanol
10 ml glacial acetic acid
Store at ambient temperature
This buffer can be made from purchased tablets (ensure the molarity and pH are correct) or more cheaply made in the laboratory.
Sodium chloride, 8.7g
Potassium dihydrogen phosphate, 0.272g
Disodium hydrogen phosphate, 1.136g
Disodium hydrogen phosphate.2H2O, 1.41g
Disodium hydrogen phosphate.12 H2O, 2.83g
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
Sodium chloride, 8.7g
Concentrated hydrochloric acid
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.
PBS (as above) containing 0.1% bovine serum albumin and 0.1% sodium azide.
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.
Hydrogen peroxide in methanol/PBS is recommended for cell preparations.
30% hydrogen peroxide, 1 ml
70% methanol in PBS, 100 ml
(0.3% hydrogen peroxide)
Make the H2O2 solution just before use. Immerse preparations for 30 minutes then rinse in PBS.
For 2 L:
Citric acid, 3.8g
2 M sodium hydroxide (8% aq)
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.
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
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.
Using a domestic microwave oven with a revolving plate
*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.
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.
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.
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.
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.