Blocking Aldehydes

The production and demonstration of aldehydes occupies an important place in histological practice. The demonstration of carbohydrates would hardly be possible without the ability to produce aldehydes by the oxidation of 1-2 glycols and their subsequent condensation with Schiff's reagent in the PAS reaction, for instance. Several other techniques depend on similar procedures as well, both with Schiff's reagent and with other means of visualising their presence.

There are, however, occasions when it may be necessary to show that the aldehydes being visualised did come from the specific treatment given and were not there prior to that, from the fixative, for instance. Similarly, it may be necessary to prove that the colouration finally obtained is due to an aldehyde and not something else, since very few techniques are inherently so specific that they will react with a single tissue component. This includes Schiff's reagent and silver reductions, although anomalous results are more likely with silver reduction than with Schiff's reagent.

Applying treatment which blocks aldehydes from taking part in reactions can overcome those difficulties.

1. Applying an aldehyde block before applying reagents to produces aldehydes can prove that the reagents did form the aldehydes responsible for a positive result.
2. If one of two adjacent sections has an aldehyde block applied after generating an aldehyde but before the visualising step,
   • If the unblocked section is coloured and the blocked section is not coloured, it proves that an aldehyde is responsible for the colour.
   • If the unblocked section is coloured and the blocked section is also coloured to the same degree, it proves that some other constuent is responsible for the colour in those areas, but that it is not an aldehyde.
   • If the unblocked section is coloured and the blocked section is also coloured but to a greater degree and in different areas, it proves that both an aldehyde and some other constuent are both responsible for the colour.

Blocking Solutions

Sulphite block
Sodium bisulphite	1.041 g
Distilled water	100 mL

Apply for 2-4 hours at 22°C. It is reversed by 2-10 minute application of 3% hydrogen peroxide, 1% ferric chloride, 1% sodium iodate or 1% potassium chlorate.

Aniline acetic block
Aniline	10 mL
Acetic acid, glacial	90 mL

Apply for 20-30 minutes at 22°C.

Aniline chloride block
Aniline	9 mL
Hydrochloric acid, conc	8 mL
Distilled water	100 mL

Shake well when adding the aniline to the acid, then dilute with water. Apply 1-6 hours at 22°C.

Phenylhydrazine block
Phenylhydrazine hydrochloride	5 mL
Acetic acid, glacial	10 mL
Distilled water	35 mL

Apply for 2-3 hours at 60°C.

Hydroxylamine block
Hydroxylamine hydrochloride	10 g
Sodium acetate	20 g
Distilled water	40 mL

Apply for 1-3 hours at 22°C. It is reversed by periodic acid and should not be used to block pre-existing aldehydes.

Semicarbazide block
Semicarbazide hydrochloride	2 g
Sodium acetate	5 g
Distilled water	40 mL

Apply for 2-3 hours at 60°C.

Procedure

1. Place into the solution for the time and at the temperature given.
2. For the sulphite block transfer directly to Schiff's reagent.
3. For the other blocks wash with running tap water for 5 minutes.
4. Rinse with distilled water.
5. Place into Schiff's reagent

 

Reference

Lillie, R.D., (1954)
Histopathologic technique and practical histochemistry Ed.2
Blakiston, New York, USA.

Pearse, A. G. E., (1968, 1972)
Histochemistry: Theoretical and Applied, Ed. 3
Churchill Livingstone, Edinburgh, London, UK