Immune fluorescence dyeing method for wall cells and suspended cells.

"Cell
Culture
" Editor-in-Chief, 2004
steps
(take indirect immunofluorescence as an example)
1 cell preparation and fixed
(1) single-layer growth cells: take the number of growth cells, with 0.25% pancreatic
protease
digestion, made of single cell suspension. Inoculate the cells into
culture bottles
or petri dishes pre-positioned with several 6×22mm cover slides, culture them in a carbon dioxide culture tank for 1-3 days, wait for the cells to grow close to a single layer, remove the coverslips, wash them into PBS (0.01 mol/L, pH7.4), and then select a necessary fixation agent to secure the cells according to the experimental purpose. Common fixants are: 95% ethanol (fixed time 10-30min), acetone (5-10min) and so on.
(2) Suspended growth cells: take the number of growth cells, wash with PBS centrifugation (1000rpm, 5min) 2 times, or use the cell centrifugal chip mechanism to prepare cell slices or directly prepare cell smears, the cell slices immersed in 95% ethanol or acetone fixed.
2 place the fixed cell slides in the cap dye cylinder, wash 5min with PBS, and remove the blow-dry.
3 drops plus appropriately diluted specific
antibody
, placed in a wet box, 37 degrees insulation 30-60min or degrees refrigerator overnight
4 PBS vibration wash 3 times, each 5min, blow-dried.
5 drops of properly diluted luciferin-labeled antibodies, placed in a wet box, 37 degrees insulation 30-60min.
6 PBS 2 times, 5min at a time, and then 1 time with distilled water.
7 50% buffer glyceer oil seal.
results
observed under a fluorescent
microscope
, the positive parts appear fluorescence, according to the type of fluorescent different colors of fluorescence, into fitC yellow-green fluorescence, Rodinming B200 orange-red fluorescence.
be observed and photographed in time after the staining reaction of the specimen. If there is no time to observe the specimen immediately, the specimen can be stored in a 4 degree refrigerator. It should be noted, however, that specific fluorescence decreases by 30% after overnight and by 50% after one week. If sealed with polyethylene alcohol, it can be stored for a long time.
.