SPI-Mark™ Colloidal Gold Reagents
Specific Immunolabeling Protocols
labeling of Cell Surface Antigens for LM
(A) Isolate cells from tissue with trypsinisation or separate blood cells by
gradient centrifugation.
(B) Wash cells in
PBS
+ 1% BSA and suspend to 10 x 109 cells / litre in PBS BSA.
(C) Prepare a cytocentrifuge preparation or make a smear from the buffy coat C
glass slide coated with BIOBOND.
(D) Air dry overnight.
(E) Fix for 30-90 sec with phosphate buffered 9.25% formol and 45%
acetone at pH 6.6.
(F) Rinse the slide in PBS.
(G) Incubate for 10 min in PBS + 5% non-fat milk or 0.01% casein + 5% normal
serum (of the same species as the gold conjugate) + required concentration of
the gold conjugate.
(H) Rinse with PBS.
(I) Post fix the cells with buffered formal acetone for 2 min at room
temperature.
(J) Wash the slides thoroughly in distilled water.
(K) Silver enhance while monitoring under the LM.
(L) Rinse with distilled water to stop enhancement.
(M) Counterstain with May-Grunwald-Giemsa stain.
(N) Mount in BIOMOUNT™ to prevent fading of the silver stain.
(O) View in the LM using bright field and epipolarised light.
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Thursday May 17, 2012
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