小中大protocol for
Immunostaining using Indirect Immunofluorescence
in Dr. Barres' lab
1) Prepare "antibody" buffer:
Distilled water 200 ml
NaCl (150 mM) 2.25 g
Tris Base (50 mM) 1.5 g
BSA (Sigma A2153) (1%) 2.0 g
L-Lysine (100 mM) 3.6 g
Azide (0.04%) Add 2 ml of a 4% stock in water
Adjust to pH 7.4 (pH paper) About 6 ml 1 M. HCl
Store at 4o C indefinitely.
2) Transfer cover slips to staining box. Gently transfer coverslips from 24-well plate to staining box (a homemade box with 24 flat topped pedestals glued to its bottom to support coverslips and keep them raised above the botom which will have some water for humidification; it should also have a top to prevent evaporation and keep slides humidified) using a coarse pair of forceps (keep cell side face up!). All volumes below are 50-100 ul unless otherwise specified, but you can use as little as 25 ul if necessary.
3) Fixation. 4% paraformaldehyde for 10 minutes (unless for BrdU staining or other fix-sensitive antigens, then use 60-90 seconds maximum). (For intracellular antigens such as GFAP can use instead a 10 minute fix in -20o C acid-alcohol (5% glacial acetic acid plus 95% ethanol) or in 100% methanol.; whenever alcohol fixation is used the triton permeabilization step can be skipped.) Paraformaldehyde cross links proteins so that they do not solubilze when the cells are permeabilized (below). By contrast, alcohol fixation works not by cross linking but by precipitating the proteins.
After the 10 minute fix step, pick up the coverslip with forceps and rinse it in three washes of PBS.
4) Block Non-Specific Binding. Incubate in 50% goat serum diluted in the "antibody" buffer for 30 minutes. The goat serum contains high levels of goat antibodies and proteins which bind non-covalently to non-specific binding sites and also bind covalently to reactive aldehyde groups created by the paraformaldehyde. Without this step, the primary antibody would bind both to these reactive aldehyde groups and the non-specific binding sites and create high levels of background staining. (Goat serum is used because the fluorochrome-conjugated secondary antibodies (see below) are raised in goats; this minimizes any unwanted binding of the secondary antibody to the goat antibodies that bind to the non-specific binding sites.)
After the block step, drain the coverslip, but it is not necessary to rinse it in PBS.
5) Permeabilization. Triton X-100 (0.4%; which is 1:50 of a 20% stock in water) for 30 minutes. This step can be combined with step #4 by adding the triton to the goat serum solution. Do not permeabilize when surface staining is being performed, particularly for galactocerebroside (a glycolipid) staining or A2B5 (a ganglioside) staining. These lipid antigens would be solublized by triton.
After permeabilization, rinse the coverslip three times in PBS (particularly for alcohol fixes).
6) Primary antibody incubation. Incubate the coverslip in the primary antibody solution for 30 minutes (or 60 minutes to overnight if necessary). To prepare this solution, dilute the primary antibody with the "antibody" buffer. The best dilution depends on the concentration of primary antibody and the density of the antigen. Generally supernatants are used neat or diluted up to 1:10. Ascites are used at 1:100 to 1:1000, as are polyclonal antisera. (The optimal dilution can be determined in a separate experiment.) Note: typical supernatant antibody concentration is 1-10 ug/ml, ascites concentrations are 1-10 mg/ml, as are polyclonal antisera. Thus typical final primary concentrations are about 1-10 ug/ml. (When the best concentration is not known and a guess must be made, I generally guess to use the supernatant undiluted, the ascites at 1:100, and polyclonals at 1:500. But beware of the "prozone" effect especially with IgM monoclonal antibodies).
