3.3.2

Generation of

Transverse Sections of

Tissue Models

1. Section the wax blocks containing the tissue models at a thick-

ness of 5–7 μm using a rotary microtome.

2. Lift the sections onto a water bath at 37 ^C and mount onto

positively charged slides.

3. Place slides onto a heated slide rack at 30 ^C and leave for a

minimum of 1 h prior to further processing.

3.3.3

Histological

Analysis

1. Deparaffinize the tissue sections in Histo-Clear for 5 min.

2. Rehydrate the samples through sequential ethanol concentra-

tions: 100% ethanol for 2 min followed by 95% ethanol, 70%

ethanol, and distilled water for 1 min each.

3. Stain the slides with Mayer’s hematoxylin for 5 min.

4. Wash slides in distilled water for 30 s.

5. Blue the nuclei with alkaline alcohol for 30 s.

6. Dehydrate the samples through 70% ethanol and 95% ethanol

for 30 s each.

7. Stain the samples with eosin for 1 min.

8. Dehydrate the samples with two washes in 95% ethanol for 10 s

each followed by two washes in 100% ethanol for 15 and 30 s

respectively.

9. Move the slides through two washes with Histo-Clear for

3 min each.

10. Mount a clean coverslip onto the slides using Omnimount.

11. Leave slides to dry at room temperature prior to imaging.

3.3.4

Immunofluorescent

Analysis

1. Deparaffinize the tissue sections in Histo-Clear for 5 min.

2. Rehydrate the slides sequentially through 100% ethanol, 70%

ethanol, and PBS for 5 min each.

3. Place the slides into citrate buffer at 95 ^C for 20 min to

perform antigen retrieval.

4. Cool the slides slowly then perform blocking and permeabili-

zation for 1 h using 100 μL of PBS containing 20% neonatal

calf serum and 0.4% Triton X-100.

5. Wash the samples three times in PBS for 5 min each.

6. Incubate the samples for 2 h at room temperature with 100 μL

of blocking solution containing the primary antibody at the

relevant dilution (Table 1).

7. Wash the samples three times in PBS for 5 min each.

8. Incubate the samples for 1 h at room temperature with 100 μL

of blocking solution containing the secondary antibody at the

relevant dilution (Table 2).

9. Wash the samples three times in PBS for 5 min each.

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