Sample type required: Deparaffinized and rehydrated tissue sectioned at 2–4 µm on positively (+) charged slides
Preferred fixative: 10% neutral buffered formalin (NBF); metal fixatives should be avoided
Control: Normal kidney
| Reagent | Time | Technical Notes |
| 1% periodic acid | 15 minutes |
|
| Rinse thoroughly in distilled water | 2 minutes
|
|
| Methenamine Silver Solution | *See technical notes | - Place slides in a plastic Coplin jar filled with working methenamine silver solution
- Add blank slides to the Coplin jar as needed to ensure equal heat distribution to the solution
- Apply lid loosely or cover loosely with gauze
- Also place a Coplin jar filled with distilled water in the microwave
- Microwave both jars for 20 seconds at 100% power
- Microwave both jars again for 20 seconds at 70% power
- Remove staining jar; allow slides to sit at rest for 5 minutes
|
| Rinse slides in heated distilled water | | |
| Tone with gold chloride | 15–30 seconds | Sections should appear brown |
| Rinse slides in distilled water | 5–6 changes | |
| 3% sodium thiosulfate | 1 minute |
|
| Rinse slides in distilled water | 5–6 changes |
|
| Counterstain | | If using an H&E counterstain, follow the H&E staining method beginning with Hematoxylin. The clarifier step should be omitted. A light green solution may also be used as a counterstain. |
Post staining procedure:
The tissue section should be rinsed well in distilled water and dehydrated through graded alcohols (beginning with 95%). Two changes of xylene should follow, followed by a coverslip.
Expected results:
• Basement Membranes - Black
• Nuclei - Blue
• Cytoplasm/Background - Pink
Limitations:
- Slides should not be handled with metal forceps or any metallic substance as a black precipitant will form from exposure to silver solution.
- Tissue must be sectioned at 2–4 µm due to the ultraminute thickness of the glomerular basement membrane. These are more readily demonstrated in thinner tissue sections.
