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Coating Slides with a Film of Nitrocellulose
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Coating Slides with a Film of Nitrocellulose

J. A. Kiernan

Department of Anatomy and Cell Biology

The University of Westen Ontario, London, Canada

Nitrocellulose is a name for several products obtained by treating cellulose with a mixture of concentrated nitric and sulphuric acids. These are not nitro compounds (which have -NO2  directly joined to carbon). They are cellulose nitrates  -  nitric acid esters in which variable numbers of cellulose C-OH groups have been changed to C-O-NO2.  Guncotton, a military explosive, is a cellulose nitrate. So is celluloid, the highly flammable material of which early 20th century movie films were made. Flexible collodion is another form, which was used to dress wounds. In histology, this useful material is commonly called celloidin, and the names parlodion, necolloidin and low-viscosity nitrocellulose (LVN) are also ecountered. Some of these words were and may still be registered trade marks. I choose to use the chemically inaccurate but tolerably short and legally uninteresting word nitrocellulose.

It probably does not matter which form of nitrocellulose is used for making films to keep sections on slides. My practical experience is limited to two types, which are intended principally for use as embedding media. These became ridiculously expensive in the 1980s. Fortunately, the solution needed for coating slides is very dilute and can be poured back into its bottle and reused many times.

When mounted sections have to be subjected to rough treatment, especially immersion in alkalis or hot acids, it is advisable to supplement the adhesive by encasing the slide in a film of nitrocellulose. This will hold the sections in place if there is failure of the adhesion between tissue and glass. The film must cover the whole slide (both surfaces and all four edges and eight corners) if it is to be effective. A nitrocellulose film is permeable to dyes and other reagents used for staining.

The procedure is as follows:

1. Take slides with attached sections, in a glass or metal staining rack, to absolute alcohol.

2. Immerse in ether-alcohol (equal volumes of ethanol and diethyl ether) for 30-60 seconds.

3. Immerse in a 0.2-0.4% solution of nitrocellulose in ether-alcohol for 30 seconds. (The solution can be made by diluting one of the stock solutions kept for nitrocellulose embedding. Solutions of nitrocellulose can be kept for years and used repeatedly. The exact concentration does not seem to matter for coating slides.)   Caution.  Ether is volatile and dangerously flammable.

4. Lift out the slides, drain, and allow them to become almost dry. A change in the reflection of light from the glass surface indicates the moment at which to move on to Stage 5. This end-point is easily learned with a little practice.

5. Place the slides in 70% ethanol for 2 minutes to harden the film of nitrocellulose.

6. Carry out the staining procedure. Dehydrate as far as the 95% alcohol stage.

7. Transfer the slides from 95% alcohol into ether-alcohol, with minimum agitation. Leave in ether-alcohol for 2-3 minutes to dissolve the nitrocellulose film and complete the dehydration. [Methanol could almost certainly be used instead of ether/ethanol for this step, but I have never tried it.]

8. Carefully take the slides from ether-alcohol into xylene (1 minute) and then into a second change of xylene (at least 1 minute). Try not to agitate the slides: this could loosen the sections. Remember!  The nitrocellulose protection was removed in Step 7. 

9. Apply coverslips, using a resinous mounting medium.

Recommended reading.

Gabe, M. 1976. Histological Techniques. Paris: Masson. pp. 142-143.   Out of print - a pity!.

Kiernan, J. A. 1999. Histological and Histochemical Methods: Theory and Practice. 3rd ed. Oxford: Butterworth-Heinemann, p. 52.  Reprinted 2000, and now published by Arnold Publishers, London - (ISBN0750649364)

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