Methenamine Silver Stain Kit For Basement Membrane (PASM) 

Basement membrane methenamine silver staining (Periodic Acid–Silver Methenamine, PASM) is a classical histochemical staining method widely used for the visualization of basement membrane structures and related mucopolysaccharide components in tissues. This method is based on the oxidative reaction of periodic acid, which oxidizes vicinal diol groups in polysaccharide structures within tissues to generate aldehyde groups, thereby providing reactive sites for the subsequent silver staining reaction.

During the staining process, gold chloride is often used for toning treatment. This step converts part of the deposited metallic silver into the more stable metallic gold, which improves staining stability and enhances structural contrast, resulting in a clearer background. Subsequently, sodium thiosulfate is used to fix the staining and remove unreduced silver ions, thereby reducing background staining and producing clearer staining results.

MCE Methenamine Silver Stain Kit For Basement Membrane (PASM) is developed based on the classical staining principle described above. Through an optimized staining system and standardized reagent combination, it enables clear visualization of basement membranes and related reticular structures in tissue sections. This method is particularly widely used in renal pathology research, where it is commonly applied to examine morphological alterations of the glomerular capillary basement membrane, such as thickening, rupture, folding, double-contour (tram-track) appearance, or abnormal proliferation caused by inflammatory injury. In addition, this method can also be applied to the histological investigation and morphological observation of glomerular diseases, diabetic nephropathy, and other basement membrane–associated pathological changes.

Periodic Acid Solution, Silver Nitrate Solution, Methenamine Solution, Gold Chloride Solution: 4°C, 6 months. Protected from light.

Ferric Ammonium Sulfate Solution, Borax Solution, Sodium Thiosulfate Solution, Light Green Solution: RT, 6 months.

Reagents Preparation

Xylene, neutral mounting medium or mounting reagent, graded ethanol solutions, distilled water, etc.

Procedure

1. Deparaffinization and Rehydration: Paraffin sections are first deparaffinized in xylene, followed by treatment with graded ethanol solutions, and then rehydrated in distilled water.

2. Periodic acid oxidation: Incubate the sections in Periodic Acid Solution for 15 min at room temperature. Rinse with running tap water, followed by washing with distilled water for 2 min.

3. Ferric Ammonium Sulfate Solution treatment: Place the sections in Ferric Ammonium Sulfate Solution for 10 min. Rinse with running tap water, then wash with distilled water for 2 min.

4. Preparation of Methenamine Silver stock solution: Prepare the methenamine silver stock solution by mixing Silver Nitrate Solution and Methenamine Solution at a ratio of 6:100 (v/v). A small amount of precipitate may appear after mixing; ensure it is fully dissolved before use.

Note: The prepared stock solution can be stored at 4°C for up to 3 months.

5. Preparation of Methenamine Silver working solution: Prepare the working solution by mixing Methenamine Silver stock solution and Borax Solution at a ratio of 106:100 (v/v). The Methenamine Silver working solution should be freshly prepared and used immediately, as it is not suitable for long-term storage. After preparation, preheat the solution in a 60°C incubator for approximately 30 min.

6. Methenamine silver staining: Place the sections in the preheated Methenamine Silver working solution and incubate in a 60°C water bath for approximately 25–30 min, until the tissue appears tobacco-brown or black. Rinse thoroughly with distilled water.

7. Toning and fixation: Immerse the sections in Gold Chloride Solution for 1–2 min for toning, then rinse with distilled water for 2 min. Subsequently treat with Sodium Thiosulfate Solution for 1 min, followed by thorough washing with running tap water.

8. Counterstaining, dehydration, and mounting: Counterstain the sections with Light Green Solution for 1 min, then rinse with tap water. Dehydrate the sections starting from 95% ethanol. Since Light Green is soluble in ethanol, slight decolorization during dehydration is normal. Light green serves mainly as a background stain, and variations in intensity generally do not affect the visualization of the basement membrane. Finally, clear the sections in xylene and mount with neutral resin or mounting medium for microscopic observation.

Interpretation of Staining Results

1. Basement membrane structures (e.g., glomerular basement membrane and glomerular capillary basement membrane): Black.

2. Tissue background: Green.

1. Glassware used in the experiment should be thoroughly soaked in a suitable cleaning solution in advance and rinsed completely. If a dedicated cleaning solution is unavailable, the glassware may be soaked in 0.1 M hydrochloric acid, thoroughly rinsed with water, and dried before use.

2. The Methenamine Silver stock solution can be stored at 4°C for up to 3 months.

3. The Methenamine Silver working solution should be freshly prepared before use and is intended for single-use only; it should not be stored for extended periods.

4. After opening, the reagents should be used as soon as possible and stored under the recommended conditions to avoid affecting subsequent experimental results.

5. During the gold chloride toning step, it is recommended to observe the color change of the sections under a microscope in real time to achieve optimal staining results.

6. This product is for R&D use only, not for drug, household, or other uses.

7. For your safety and health, please wear a lab coat and disposable gloves to operate.