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Hillebrands1 project protocol

Aging study: Lymphocytic infiltration in kidneys of 23 inbred strains of mice, age 20 months   (2014)

Hillebrands J, Korstanje R
With: Huang Y, Caputo CR, Noordmans GA, Yazdani S, Monteiro LH, van den Born J, van Goor H, Heeringa P




Hillebrands1 Protocol

Project protocol - Contents

Workflow and sampling

Step
Procedure
 Equipment
Data collected
1 Mice euthanized at 20 months of age, kidneys harvested Dissection kit -
2
 Kidney fixed, sectioned, stained, analyzed Microscope
Number of perivascular clusters per kidney section, cumulative size of perivascular clusters

Equipment and supplies

  • Dissection kit
  • Microscope
  • Computer for morphometric analysis

Reagents and solutions

  • Bouin's fixative
  • Paraffin
  • Periodic acid-Schiff staining reagent

Procedure: Kidney histopathology

    1. Kidney tissue is fixed in Bouin's fixative followed by embedding in paraffin.
    2. Paraffin-embedded blocks are cut into 2 µm sections and Periodic acid-Schiff staining (PAS) is performed.
    3. PAS staining is used for computerized morphometric analysis.
    4. Because the size of immune cell clusters depends on the way the tissue is cut and their localization, clusters are measured as follows:
      • Perivascular clusters found around blood vessels at the renal hilum area are excluded
      • Total number of perivascular clusters per kidney section as well as the total cumulative size of the clusters are determined.

Data collected by investigator

  • total number of perivascular clusters per kidney section
  • total cumulative size of perivascular clusters [relative cluster size = (total cumulative cluster area/total renal tissue area) x 100]