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The page below is a sample from the LabCE course Histology Special Stains: Connective Tissue. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Jones' Methenamine Silver Stain (JMS) - Staining protocol

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

ReagentTimeTechnical Notes
1% periodic acid15 minutes
Rinse thoroghly in distilled water2 minutes

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 thiosulfate1 minute
Rinse slides in distilled water5 -6 changes
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.