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 4oC 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.
