Histology information
Decalcification
Any bony tissue (paws, osteochondral plugs, tibial plateaux etc) will need decalcifying prior to sectioning or they could shatter and not cut properly. This can be done relatively rapidly with formic acid but with the caveat that this acid may destroy certain antigens for IHC. EDTA is much gentler method of decalcification that preserves most antigens but takes about twice as long as formic acid. A mouse knee joint takes 2 weeks to decalcify in formic acid and 4 weeks in EDTA.
If you are unlucky enough to want to detect an antigen that is affected by decalcification then you may be able to cryosection the sample using a tungsten carbide blade but you will have to cut very thick sections and use a confocal microscope.
Stains
Which Stain to choose depends on what structure you want to see (apart from the pretty colours!) in order to score any pathology present. Haematoxilyn & Eosin and Safranin-O & Fast green are the two most common stains we perform at the Kennedy but we are competent at the using the following stains:
Giemsa
Helicobactor (in gut), less differentiated intestinal crypt cells stain dark purplish blue while basement membrane stains light blue and goblet cells purple. Also differentiates nuclei and cytoplasm.
Haematoxilyn & Eosin
Nuclei dark blue while all other structures are various shades of pink and purple, very good general stain for quantifying inflammation as nuclei are obvious and differentiation of granulocytes possible. If you are not sure, try H&E first.
Massons Trichrome
For detecting fibrosis and distinguishing cells from connective tissue. Keratin and muscle are red, collagen & bone are are blue/green and cytoplasm is light pink.
Picrosirius red
Differentiates between type 1 and type 2 collagen when viewed under polarised light and very good for highlighting the basement membrane in the gut.
Safranin-O & Fast Green
Safranin-O will stain cartilage red in proportion to its proteoglycan content and fast green stains bone green. Coupled with a haematoxylin nuclei counterstain, Safranin-O/Fast green is the standard stain for assessing osteoarthritis.
There are many other stains and kits in the histology cupboard, so have a look before buying something.
