Sample type required: Deparaffinized and rehydrated tissue sectioned at 2 - 4µm 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 thoroghly in distilled water | 2 minutes
|
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| Methenamine Silver Solution | *see technical notes | - Place slides plastic coplin jar filled with working methenamine silver solution.
- Add blank slides to coplin jar as needed to ensure equal distribution of heat to solution.
- Apply lid loosely or cover loosely with gauze.
- Also place coplin jar filled with distilled water in 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 - 30seconds | 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 |
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| Counterstain | | If using H&E counterstain, follow H&E staining method beginning with Hematoxylin. Clarifier step should be omitted. Light green solution may also be used as a counterstain. |
Post staining procedure:
Tissue section should be rinsed well in distilled water, dehydrated through graded alcohols(beginning with 95%). Follow with two changes of xylene and then 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 2-4µm due to the ultraminute thickness of glomerular basement membrane. These are more readily demonstrated in thinner tissue sections.
