Untitled

Histology of Adipose Tissue – MacDougald lab 2014

During dissections, a standard necropsy procedure should be maintained between animals to minimize any variance in tissue collection. A wet-tissue weight is taken immediately after removal using an analytical balance. Special care is taken to remove any nonadipose-associated tissues from the depot including glands and lymph nodes.

Once weighed, 100–500 mg of the adipose tissue of interest is placed in a sealable tube and covered by a ratio of >10 mL of 10% formalin per gram of WAT (Sigma-Aldrich, Cat #HT501320). Covered tissue is placed at 4^oC for 72 h, then switched to 70% ethanol (equal volume as was used for formalin) for 48 h. While extended formalin storage can result in difficulty with certain antigen detection due to “overfixation”, this has little impact on the current method as buffered formalin will not cause variable tissue hardening (Fox, Johnson, Whiting, & Roller, 1985). Of greater concern is the degradation of the tissue as a result of under-fixation and fixative procedures that lead to variable tissue “shrink- age.” A standard procedure should therefore be strictly followed for WAT fixing and dehydration.

1. Identify adipose tissue depot of interest

2. Carefully dissect out adipose depot removing nonadipose-associated tissue (e.g., glands, lymph nodes)

3. Weigh depot

4. Cover 100–500 mg of tissue with >10-fold volume (mL) of 10% formalin to tissue (g) and let sit at 4 ^oC for a minimum of 72 h

5. Remove tissue and place in 70% ethanol for 48 h

2.1.2 Tissue embedding

Preserved tissues are then placed in labeled histology cassettes (e.g., Cat# CA95029-822, CA18000-174; VWR International, Radnor, PA, USA) and paraffin-processed (e.g., Leica ASP 300 Paraffin Tissue Processor, Leica Microbiosystem, Wetzlar, Germany). Below are the steps used for paraffin processing adipose tissue.

1. 1 h in 70% ethanol

2. 1 h in 80% ethanol

3. 1 h in 95% ethanol (x2)

4. 1 h in 100% ethanol (x2)

5. 1 h in xylene (x2)

6. 1 h in 60 ^oC paraffin (x3)

Once the sample is processed, the tissue is embedded into a paraffin block (e.g., Tissue-Tek Paraffin Tissue Embedder, Sakura Finetek, Torrance, CA, USA) and stored at 4 ^oC. Time and care should be taken in the embedding process to ensure the tissue is placed into the center of the block. This will help standardize each sample for sectioning.

Note: Allowing the tissues to remain in the 60 ^oC paraffin for long periods of time after processing should also be avoided to limit tissue damage.

2.1.3 Sectioning, deparaffinizing, and staining adipose tissue

Embedded tissue samples are faced off using a low profile microtome blade (e.g., Accu-Edge, Sakura Finetek, Cat# 4689, Sakura Finetek, Torrance, CA, USA) to the apex of the tissue using a paraffin microtome (e.g., Leica 2155 rotary paraffin microtome, Leica microbiosystems, Wetzlar, Germany).

Note: Samples should be kept on ice between sectioning. Colder blocks

will provide cleaner sections. Similarly, a 40–42 ^oC water bath should be used to collect sections. When the dehydrated tissue comes into contact with water the tissue will begin to expand. Embedded adipocytes are fragile. Therefore, if the water is too warm the tissue will expand quickly tearing the delicate adipocyte membrane and leading to difficulties with downstream analysis.

Starting at the tissue apex 5 X 5 mm sections are made at a minimum of 100 mm intervals across the sample tissue. Using a minimum interval distance of 100 mm will ensure that each section will contain a unique sampling of adipocytes. Serial sections are recommended as this will provide matching tissue samples for additional independent analyses (e.g., immuno-histochemistry). Sections are placed onto labeled electrostatically charged precleaned microscope slides (e.g., Cat# 12-550-15, Thermo Fisher Scientific, Waltham, MA, USA) and are left to dry for a minimum of 24 h.

1. Embedded tissues are faced off to tissue apex

2. 5 X 5 mm sections are taken at 100 mm intervals and placed on slides

3. Slides are allowed to dry for 24 h

Note: Cutting tissue seems like an inconsequential process, however paraffin-embedded adipose tissue is very delicate and membranes are prone to tearing. Extreme caution should be taken with sectioning. It is wise to perfect sectioning on nonessential adipose tissue samples before moving onto important samples.

Once dry, slides are placed into a 60 ^oC dry oven for 1.5 h to begin removal of paraffin, and any residual water. Tissues are then stained with H&E using the following method.

1. Slides are baked at 60 ^oC for 1.5 h

2. 5 min in xylene (X2)

3. 2 min in 100% ethanol (X2)

4. 2 min in 95% ethanol (X2)

5. 2 min in 70% ethanol

6. 2 min in deionized H₂O

7. Stain for 2 min in Harris’ hematoxylin (Cat# HHS16, Sigma-Aldrich, St. Louis, MO, USA)

8. Rinse under running H₂O until clear

9. Three dips in a bluing solution

10. Rinse for 2 min under gently running H₂O

11. 2 min in 80% ethanol

12. 2 min in eosin (Cat# 318906, Sigma-Aldrich, St. Louis, MO, USA)

13. Four dips in 80% ethanol to rinse out excess eosin

14. 2 min in 95% ethanol

15. 2 min in 95% ethanol

16. 2 min in 100% ethanol (x2)

17. 3 min in xylene (x3)

Slides are then treated with a xylene-based permount and coverslips mounted (e.g., Cat# 2955-245, Corning Incorporated, Corning, NY, USA). Slides should be left horizontal to set for at least 24 h before putting them into a storage box.