Histology

Immunohistochemistry protocol for F4/80 staining of formalin fixed paraffin embedded gWAT. 

Hunter, A. L., Pelekanou, C. E., Barron, N. J., Northeast, R. C., Grudzien, M., Adamson, A., Downton, P., Cornfield, T., Cunningham, P. S., Billaud, J-N., Hodson, L., Loudon, A. S. I., Unwin, R. D., Iqbal, M., Ray, D. W., Bechtold, D. A (2021). Adipocyte NR1D1 dictates adipose tissue expansion during obesity. eLife 2021;10:e63324 DOI: 10.7554/eLife.63324.

Table of Reagents

Equipment/Consumables

Gonadal (gWAT) adipose collection, fixation and processing

1. Fix gonadal (gWAT) adipose in 4% paraformaldehyde (PFA) at 4°C for 24hrs. 
2. Discard 4% PFA and replace with 70% Ethanol or IMS. Incubate at 4°C for 24hrs.
3. Transfer adipose to pre-labelled cassettes and process using the ‘routine overnight’ setting on the Leica ASP300s.
4. Embed the adipose in paraffin wax and leave to set. 
5. Cut 5µm sections using a Leica RM2255. Cut ribbons and float sections onto the surface of Superfrost^™Plus Adhesion microscope slides. 
6. Leave sections to dry at 40°C in a LEEC drying cabinet for 12-15 hours. 
7. Store cut sections at room temperature. 

Immunohistochemistry staining protocol

Day 1;

1. Deparaffinization and hydration of 5µm formalin-fixed, paraffin-embedded (FFPE) sections (perform at room temperature in a fume hood):
   1. Incubate sections in two changes of xylene for 5 minutes each.
   2. Incubate sections in two changes of 100% IMS for 5 minutes each.
   3. Incubate sections in 70% IMS for 1 minute.
   4. Incubate sections in 50% IMS for 1 minute.
   5. Wash sections in two changes of dH₂O for 5 minutes each. Do not allow sections to dry out following deparaffinization and hydration.
2. To perform antigen retrieval, prepare the enzymatic antigen retrieval solution by dissolving 1 trypsin tablet in 1mL PBS. Vortex to mix well.
3. Using a pipette, add 100-200µL of trypsin/PBS antigen retrieval solution to each section and incubate in a humidified dark chamber for 10 minutes at room temperature.
4. Wash sections in three changes of dH₂O for 5 minutes each.
5. Following the last wash, tap off excess dH₂O on to absorbant paper towel. Using a liquid blocker super pap pen, draw around the section.
6. Transfer the slides to a humidified dark chamber and add 100-200µL 3% hydrogen peroxide/dH₂O (H₂O₂) solution to each section. Incubate for 30 minutes at room temperature.
7. Wash the sections in two changes of dH₂O for 5 minutes each.
8. Wash the sections in PBS + 0.1% Tween-20 for 5 minutes.
9. Transfer the slides to a humidified dark chamber and using a pipette add 100-200µL of blocking solution (5% normal goat serum diluted in PBS +0.1% Tween-20). Incubate for 1 hour at room temperature.
10. Remove the blocking solution by tapping off the excess on to absorbant paper towel. Using a pipette add 100-200µL of rat monoclonal F4/80 antibody diluted at 1:500 in PBS +0.1% Tween-20.
11. Incubate sections overnight at 4°C in the humidified dark chamber.

Day 2;

1. Remove the primary antibody solution and wash the sections with three changes of PBS +0.1% Tween-20 for 5 minutes each.
2. Transfer the slides to a humidified dark chamber. Using a pipette, add 100-200µL of biotinylated anti-goat IgG secondary antibody diluted 1:1500 in 5% goat serum diluted in PBS +0.1% Tween-20. Incubate the sections for 1 hour at room temperature.
3. Wash the sections in three changes of PBS + 0.1% Tween-20 for 5 minutes each.
4. Using a pipette, add 100-200µL VECTASTAIN® Elite® ABC kit, peroxidase (standard) to each section. Incubate for 30 minutes at RT in a humidified dark chamber. Please note the ABC reagent must be prepared 30 minutes before use as per suppliers instructions;
   1. 5 mL PBS (no Tween-20)
   2. 2 drops reagent A
   3. 2 drops reagent B
5. Wash the sections in two changes of PBS +0.1% Tween-20 for 5 minutes each.
6. Prepare the DAB substrate kit, peroxidase as per suppliers instructions;
   1. 5 mL dH₂O
   2. 2 drops buffer, mix well
   3. 4 drops DAB, mix well
   4. 2 drops H₂O₂, mix well.
7. Using a pipette, add 100-200µL of the prepared DAB substrate to each section. Monitor the progress of the reaction on an initial test slide. Stain all other slides to match reaction timing (typically 5-10 minutes).
8. Wash the sections in two changes of tap water to stop the DAB reaction.
9. If desired, counterstain the sections with haematoxylin (typically 30 seconds).
10. Wash the sections in two changes of dH₂O for 5 minutes each.
11. Dehydrate, clear and mount the sections (perform at room temperature in a fume hood);
    1. Incubate the sections in 50% IMS for 1 minute.
    2. Incubate the sections in 70% IMS for 1 minute.
    3. Incubate the sections in two changes of 100% IMS for 2 minutes each.
    4. Incubate the sections in two changes of xylene for 2 minutes each.
    5. Mount the sections using DPX, coverslip, and leave to dry.

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