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TISSUE PREPARATION:
A. CYTOSPINS:
cells are suspended in media to a concentration ofabout 1 X 106 cells/ml. Using PLL-coated, silane-activated slides, cytospin 50 ul of cells per slide. Add 50 ul serum to each cytospin chamber as a chaser prior to spinning. Air dry cells, then promptly fix for 20 minutes in PLPG fixative (freshly made). Then, wash X 3 in H20. The slides are then passed through graded alcohols to dehydrate (70% X 2, 100% X 2) and stored at700C.
B. FROZEN SECTIONS OF TISSUE:
tissue is to be frozen immediately upon receipt (from biopsy, autopsy) in liquid nitrogen, or can be directly placed onto a chuck and frozen on OCT compound on dry ice. These chucks are then given to Mr. Hap Soban, (American Histolabs) along with prepared, activated slides, for sectioning. Procedures should be done with gloves. After sectioning, they can be stored indefinitely at -70 C, and fixed later, or fixed in PLPG, and then stored at -700C until use.
C. PARAFFIN SECTIONS:
Fresh tissue can be fixed in PLPG fixative directly and placed in paraffin blocks, or you can use standardly prepared specimens in paraffin blocks ( usually fixed in 4% paraformaldehyde). PLPG fixative yields a better signal, but both should work. Tissue paraffin blocks obtained from elsewhere can be sectioned directly onto the PLL-coated, silanated and activated slides. When ready to use slides, first bake slides at 55-600C for one hour, and then deparaffinize by placing slides in xylene f or 5 minutes X 2. Then pass through two washes of 100% ETOH. They can be stored in the refigeratopr in this state indefinitely.
D.PLPG Fixative (Paraformaldehyde-Lysine-Periodate-Glutaraldehyde):
l. Buffered lysine (O.lM lysine in 0.05 M Na2PO4)
18.3 g lysine monohydrochloride
13.4 9 Na2HP04.7H20 ( or 7.1 g anhydrous)
1 liter of d2H20
pH solution to 7.4
2. 4% paraformaldehyde (should be made fresh q 2 weeks)
20 g paraformaldehyde powder 500 ml d2H20
Heat on hot plate at 60 C until partially dissolved. Add 1ON NAOH dropwise, until solution clears.
3. 8% glutaraldehyde
Polyscience EM grade glutaraldehyde (Catalog #00216) comes as an 8% solution
TO PREPARE THE FINAL PLPG FIXATIVE MIXTURE:
Mix 120 ml-of buffered lysine
20 ml paraformaldehyde solution
20 ml glutaraldehyde (2 10 ml 8% vials) 320 mg Na m-Periodate (Sigma)
Mix with stirring bar until dissolved; Keep on ice 10 minutes bef ore use. Sterilize through 45 micron filter before use.
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