Protocol for immunohistochemistry (ABC method)

1. Deparaffination and rehydration of sections by immersion twice for 5 min. each time in Rotihisto^® (Roth, Karlsruhe, Germany), once in isopropanol for 5 min.

and in a series of descending alcohols (96%, 70%, and 50% ethanol) each for 5 min.

2. Incubation for 30 min. at room temperature in H2O2 0.5% (v/v) in methanol to block endogenous peroxidase activities

3. Rinsing in 3 changes of distilled water for 5 min., then once in phosphate-buffered saline (PBS; pH 7.1)

4. Tissue pretreatment depending on the primary antibody used (see 3.4.2.1) (optional)

5. Rinsing in PBS for 5 min.

6. Insert slides in Shandon Racks (Coverplates^TM Sequenza^®, Pittsburgh, USA) 7. Incubation with blocking serum at room temperature for 30 min.

8. Incubation with primary antibody over night at 4°C in the refrigerator

9. Rinsing in 3 changes of distilled water for 5 min., then once in PBS (pH 7.1) 10. Incubation with secondary antibody for 30 min. at room temperature

11. Rinsing in 3 changes of distilled water for 5 min., then once in PBS (pH 7.1) 12. Incubation with ABC for 30 min. at room temperature

13. Rinsing in 3 changes of distilled water for 5 min., then once in PBS (pH 7.1) 14. Incubation in 0.05% (w/v) fresh and filtrated 3,

3´-diaminobenzidine-tetrahydrochloride (DAB; Fluka^®, Buchs, Switzerland) for 10 min at room temperature and under constant stirring with H2O2 0.03% (v/v) as substrate in 0.1 M Tris-buffered saline (TBS; Tris-hydroxymethyl-aminomethane; Merck, Darmstadt, Germany), pH 7.6

15. Rinsing in 3 changes of distilled water for 5 min., then once in TBS

16. Rinsing in 3 changes of distilled water for 5 min.

17. Counterstaining with Mayer’s hematoxylin, duration according to desired color intensity

18. Rinsing in 3 changes of distilled water for 5 min.

19. Dehydration twice for 2 min. through a series of ascending alcohols (50%, 70%, and 96% ethanol), once in isopropanol, clearing in EBE^® “acetic acid-n-butylester” (Roth, Karlsruhe, Germany), and finally automatic mounting (promountes^® RCM2000, Medite, Burgdorf, Germany)

3.4.2.1 Tissue pretreatment Microwave treatment

Tissue sections were immersed in citrate buffer (pH 6.0) and incubated in a microwave oven (800 Watt) for 20 min. Subsequently sections were left at room temperature for 10 min. for cooling.

Target Unmasking Fluid^® (TUF)

TUF (Kreatech Diagnostics, Amsterdam, The Netherlands) was diluted 1:4 with destilled water. The used solution was preheated in a water bath at 95°C. Tissue sections were incubated for 15 min. at 95°C. Subsequently sections were left in the cuvette at room temperature for 15 min. for cooling (KOVACEVIC et al., 1997).

Triton-X

Tissue sections were immersed in PBS with 0.5 ml (0.25%; Triton^® X-100, Serva, Heidelberg, Germany) at room temperature and under constant stirring for 15 min.

Enzymatic treatment

Tissue sections were immersed in a solution of 200 μg/ml Proteinase K (Chemicon, CA, USA, Catalog no. 21627) in PBS for 10 min. at room temperature.

3.4.2.2 Glial fibrillary acidic protein

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein of 52 kDa found in astrocytes of the CNS. In the peripheral nervous system, GFAP is

expressed in Schwann cells, enteric glial cells and satellite cells (JESSEN et al., 1984).

The antibody was raised in rabbits against bovine GFAP (DakoCytomation, Hamburg, Germany, catalog # Z334). As negative control the primary antibody was replaced by normal rabbit serum diluted 1 : 2000 in PBS. Astrocytes of the brains served as internal positive control.

The numbers of sections, stained with GFAP are listed in Table 3.2, 3.3, and 3.4.

3.4.2.3 Bandeiraea simplicifolia-immunohistochemistry

A lectin from Bandeiraea simplicifolia (BS-1; Sigma, L-3759) binds to microglia/macrophages (STREIT and KREUTZBERG, 1987). As negative control BS-1 was replaced by PBS. As positive control a canine brain with a granulomatous meningoencephalitis (S 81/04) was used. The numbers of sections stained with BS-1 are listed in Table 3.2, 3.3, and 3.4.

3.4.2.4 TUNEL-assay

The terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridinetriphosphate [dUTP] nick end-labeling (TUNEL)-assay (ApopTag^® Plus Peroxidase in situ Apoptosis Detection Kit, Chemicon Int., Hofheim, Germany) was used to identify structural changes of the deoxyribonucleic acid (DNA) during apoptosis. The DNA in cells undergoing apoptotic cell death is cleaved into fragments of 180 to 200 base pairs. For the “in situ end labeling” to detect DNA-double strand nicks the enzyme

“terminal transferase” (terminal deoxynucleotidyl transferase [TdT]) incorporates labeled nucleotides at the 3´-hydroxyl end of the DNA fragments. The incorporated nucleotides were detected subsequently by a peroxidase-labeled anti-digoxigenin antibody. The immunologic reaction was visualized by addition of a chromogen (3, 3´-diaminobenzidine-tetrahydrochloride) and hydrogen peroxide as electron donor.

As negative control for the TUNEL-assay, the TdT enzyme was omitted. As positive control a tissue section of rat mammary tissue provided by the manufacturer was used. Apoptotic cells were identified by a medium to dark brown nuclear and cytoplasmic immunostaining associated with a nuclear morphology as described for apoptotic cells (KUMAR et al., 2004; MYERS and McGAVIN, 2007). The numbers of sections, stained with the TUNEL-assay are listed in Table 3.2, 3.3, and 3.4.

TUNEL-assay protocol

1. Deparaffination and rehydration of sections by immersion twice for 5 min. in Rotihisto^® (Roth, Karlsruhe, Germany), once in isopropanol for 5 min. and in a series of descending alcohols (96%, 70%, and 50% ethanol) for 5 min.

2. Rinsing in PBS (pH 7.1) for 5 min.

3. Antigen retrieval with proteinase K (see 3.4.2.1) for 10 min. at room temperature

4. Rinsing in 4 changes of distilled water for 2 min.

5. Incubation for 30 min. at room temperature into H2O2 2.0% (v/v) in Tris-PBS, to block endogenous peroxidase activities

6. Rinsing twice in 0.1 M Tris-buffered saline (Tris-PBS; Tris-hydroxymethyl-aminomethane; Merck, Darmstadt, Germany; pH 7.6) for 5 min.

7. Placing sections in a moist chamber and incubation with equilibration buffer (supplied by the manufacturer) for 10 min. at room temperature

8. After draining, slides were placed in a moist chamber, which was warmed up at 60°C, and subsequently the Working Strength TdT solution was applied for 60 min. at 37°C

9. Immersion into stop-and-wash-buffer (supplied by the manufacturer) for 30 min. at 37°C under constant stirring

10. Rinsing in 3 changes of Tris-PBS, for 5 min.

11. After draining, incubation with anti-digoxigenin antibody for 30 min. at room temperature

12. Rinsing in 3 changes of Tris-PBS, for 5 min.

13. Incubation with the chromogen and substrate solution for 5 min. at room temperature. The chromogen used was 3, 3´-diaminobenzidine-tetrahydrochloride (Fluka, Buchs, Switzerland) 0.05% (w/v) with H2O2 0.03%

(v/v) as substrate in 0.1 M Tris-buffered saline pH 7.6.

14. Counter staining with Mayer’s hematoxylin, duration according to desired color intensity

15. Rinsing in 3 changes of distilled water for 5 min.

16. Dehydration twice for 2 min. through an series of ascending alcohols (50%, 70%, and 96% ethanol), once in isopropanol, clearing in EBE^® “acetic acid-n-butylester” (Roth, Karlsruhe, Germany), and finally automatic mounting (promountes^® RCM2000, Medite, Burgdorf, Germany).