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100% ethanol (fresh) 1:1 ethanol/xylene Xylene, several changes

5 min

The tissue should be dehydrated and embedded on the same day if possible. Leave in 70% ethanol for as short a time as possible, as long periods of storage in 70% ethanol may extract some insulin from the P granules and result in a weaker stain. The exocrine portion of the mouse pancreas may section with some difficulty if stored in Bouin's fixative for extended periods.

6. Embed tissue in paraffin, prepare 5-|m sections, and mount on glass microscope slides.

This step is usually performed in a dedicated histology facility. Zeller (1989) describes the details of paraffin embedding and mounting of fixed tissues.

7. Rehydrate paraffin sections on slides by passing through the following series:

Xylene

1:1 ethanol/xylene 100% ethanol 100% ethanol 100% ethanol 95% ethanol 70% ethanol Distilled H2O

1 min 5 min 3 min

2 min quickly quickly rinse.

8. Place slides in fresh 70% ethanol again for 2 min. Stain P granules with aldehyde fuchsin

9. Stain slides by immersing 10 to 13 min in filtered aldehyde fuchsin stain.

10. Wash stained slides in 2 to 3 changes of 95% ethanol as follows:

Wash 1 30 sec to 1 min

Wash 2 2 to 3 min

Wash 3 3 to 5 min (optional; if there are many slides).

11. Wash slides briefly in 70% ethanol, then briefly in distilled water.

Stain sections with hematoxylin/eosin and dehydrate

12. Immerse slides in Mayer's hematoxylin for 15 sec (or longer as determined empirically with multiple sections, depending on strength of hematoxylin).

13. Wash slides in gently running tap water until they turn a navy-bluish color, then rinse in distilled water.

14. Dehydrate and stain slides with eosin by passing through the following series:

50% ethanol

quickly

1% eosin Y in 80% ethanol

usually 15 to

95% ethanol

quickly

100% ethanol

2 min

100% ethanol

3 min

100% ethanol

5 min

1:1 ethanol/xylene

1 to 1.5 min

Xylene, several changes

5 min each.

Examine slides microscopically, score for insulitis infiltration, and calculate insulitis index

15. Mount coverslips on sections with mounting medium (e.g., Permount, HSR, or Coverbond). Examine microscopically.

A mouse islet with a normal complement of darkly staining (navy-blue color) p cells is shown in Figure 15.9.2A.

P cells will stain a deep purple color if well granulated or a weaker blue if partially degranulated. Any mast cells in the pancreas will also be stained purple, as will the elastin lining of capillaries and arteries.

The advantage of using the aldehyde fuchsin histochemical stain for insulin is that immunocytochemistry (unit21.4) using standard immunocytochemicalprotocols (to detect other islet cell peptide hormones or any antigen stable after Bouin's fixation/paraffin embedding) can be applied on the same slide. This is done at the distilled water step of the rehydration process (step 7) before staining with aldehyde fuchsin.

16. Score all pancreatic islets observed in at least three nonoverlapping sections per pancreas (include at least 30 to 40 islets) for the extent of insulitic infiltration as follows:

No infiltration grade 0 Perivascular/periductular infiltrates with leukocytes touching islet perimeters, but not penetrating grade 1

Leukocytic penetration of up to 25% of islet mass grade 2

Leukocytic penetration of up to 75% of islet mass grade 3

End-stage insulitis, <20% of islet mass remaining grade 4

Several grades of insulitis in the NOD pancreas using the aldehyde fuchsin stain to identify granulated P cell mass are illustrated in Figure 15.9.2.

17. Calculate insulitis index (I) according to the following formula:

I_ (0xn0) + (1 xn1) + (2xn2) + (3xn3) + (4xn4) 4 x (n0 + n1 + n2 + n3 + n4)

Figure 15.9.2 Photomicrographs (156x) of aldehyde fuchsin-stained pancreatic sections in NOD mice. (A) Insulitis-free islet in pancreas of NOD-scid/scid mouse stained with aldehyde fuchsin to identify granulated p cells (dark color). (B) Islet in pancreas of 8-week-old NOD/Lt-+/+ female showing stage 2 insulitis at one pole of islet (arrow). It is scored as "2" because leukocytes are clearly penetrating into the islet. If there were no leukocytic penetration, the score would be "1" ("perinsulitis"). (C) Islet in same pancreas as (B), but showing stage 3 insulitis. The original boundaries of this large islet are now filled in with infiltrating leukocytes, producing erosion of the P cell (dark staining) mass. An "end-stage" islet (4.0 score) would contain even fewer, if any, granulated p cells.

Figure 15.9.2 Photomicrographs (156x) of aldehyde fuchsin-stained pancreatic sections in NOD mice. (A) Insulitis-free islet in pancreas of NOD-scid/scid mouse stained with aldehyde fuchsin to identify granulated p cells (dark color). (B) Islet in pancreas of 8-week-old NOD/Lt-+/+ female showing stage 2 insulitis at one pole of islet (arrow). It is scored as "2" because leukocytes are clearly penetrating into the islet. If there were no leukocytic penetration, the score would be "1" ("perinsulitis"). (C) Islet in same pancreas as (B), but showing stage 3 insulitis. The original boundaries of this large islet are now filled in with infiltrating leukocytes, producing erosion of the P cell (dark staining) mass. An "end-stage" islet (4.0 score) would contain even fewer, if any, granulated p cells.

Animal Models for Autoimmune and

Inflammatory Disease

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