Jones' Impregnation
for Basement Membranes

Solutions

Periodic acid, 0.5% aqu.. Neutral red, 1% aqu.
Yellow gold chloride, 0.2% aqu. Light green SFy, 0.2% in 0.2% acetic acid, or
Sodium thiosulphate, 2.5% aqu. Progressive hemalum and eosin
 
Stock Methenamine silver
Methenamine, 3% aqueous 100 mL Shake until the precipitate redissolves.
Silvering of the container indicates deterioration.
Silver nitrate, 5% aqu. 5 mL
 
Working Methenamine silver
Stock Methenamine silver 50 mL Make just before use and preheat to 50°C.
Borax, 5% aqu. 5 mL

Tissue sample
3µ paraffin sections of neutral buffered formalin or Bouin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended. Thinner sections are to be preferred. This method gives excellent results with deplasticised methyl methacrylate sections at 1µ.

Method

  1. Bring sections to water via xylene and ethanol.
  2. Oxidise with 0.5% periodic acid for 15 minute.
  3. Rinse well with tap water.
  4. Rinse with distilled water.
  5. Treat with methenamine silver solution at 50°C. until impregnated (up to 3 hours)
  6. Wash with distilled water.
  7. Tone with 0.2% gold chloride solution for 2 minutes.
  8. Rinse with distilled water.
  9. Fix in 2.5% sodium thiosulphate for 3 minute.
  10. Wash well with running tap water.
  11. Counterstain with light green, neutral red or a light H&E..
  12. Rinse with tap water.
  13. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected results

Notes

  1. In order to see the basement membranes on edge it is necessary to use the thinnest sections possible, especially for glomeruli.
  2. Methenamine is also known as hexamethylenetetramine and hexamine.
  3. Borax is sodium tetraborate.
  4. Toning is a variable step. Untoned sections give dark brown material on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black material on a pale grey-brown background. Others tone longer (a few minutes) to produce black material on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.
  5. This method depends on a similar principle to the periodic acid Schiff reaction, but in which the aldehydes produced by oxidation reduce a silver solution instead of combining with Schiff's reagent to form a red compound. Consequently, those materials which are red in a PAS will be black in Jones' stain, i.e. it is not specific for basement membranes but will demonstrate any carbohydrates which can be oxidised to aldehydes.
  6. Hayashi, Tome and Shimosato recommended that, after oxidation with periodic acid, thiosemicarbazide should be applied to the section. Thiosemicarbazide has the formula H2NNHCSNH2. The hydrazine group (H2NNH-) combines with any aldehydes generated by periodic acid oxidation. The thiocarbamyl group (-CSNH2) is a more powerful reducing agent than the aldehydes it replaces and reduces the methenamine silver solution more rapidly and with higher contrast.
    Immediately following step 3:
    a. Place sections in 1% aqueous thiosemicarbazide for 10 minutes.
    b. Wash well with tap water, and carry on from step 4.
  7. It is well known that metallic azides can be explosive. However, thiosemicarbazide is not a simple metallic azide. The MSDS from J. T. Baker (via the SIRI database) says:
    Flash Point: N/A
    Lower Explosive Limit: N/A
    Upper Explosive Limit: N/A
    Unusal Fire and Expl.rds: NONE IDENTIFIED

 

Reference

Wikipedia has an article on methenamine.

Drury, R A, and Wallington, E A, (1967).
Carleton's histological technique., Ed. 5.
Oxford University Press, London, England.

Hayashi, I., Tome, Y. and Shimosato, Y., (1989)
Thiosemicarbazide used after periodic acid makes methenamine silver staining of renal glomerular basement membranes faster and cleaner.
Stain Technology, v 64, p 185.

 


 

 

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