Immunostaining for GABAergic synapses

11/29/05

Generally we have taken neurons grown on glial-coated coverslips, transfected with RNAi and GFP at DIV 4-6 then fed 2X weekly until DIV 12-14.

Recheck GFP before fixing to see that there are healthy expressing cells.

For live cell-surface GABA-A receptor labeleing, dilite anti-GABA A beta2/3 subunit antibody (Chemicon MAB341) 1:100 in NB-B27 taken from the culture dish. You will need 50uL per coverslip. Always do at least two coverslips per staining condition if possible.

Make a humidified chamber by lining the bottom of a petri dish with a damp paper towel topped with a piece of parafilm. Label the parafilm with the name of each coverslip (mef2 RNAi, vector RNAi, etc).

Remove the coverslips from the culture dish with tweezers and place face up in the humidified chamber. Pipette 50uL of the antibody solution gently on top of each coverslip. Work as quickly as possible so that the coverslips do not dry out. Transfer a single coverslip, add the antibody solution, then do the next coverslip, etc.

Return the chamber to the tissue culture incubator for 1 hour.

Move the coverslips to a 12 well dish filled with PBS. Wash 3 X 5’ with 1mL PBS/well. When you change the solution, aspirate off 1 well, add fresh PBS, then do the next well, add more PBS, etc.

Take all coverslips to be fixed into the 12 well dish and fix in 1mL 4% paraformaldehyde (pf) for 10’ at RT.

Remove the pf and store for proper disposal (this CANNOT go down the sink. Must be collected and disposed of as hazardous material).

Wash the cells with PBS 5’.

Block/permeabilize for 60’ in PBS with 16% goat serum and 0.2% Triton X-100.

Make dilutions of primary antibodies, 50uL/CS.

1) For GABAergic synapses (put on cells that already had anti-GABA A R)

Guinea pig anti-GAT1 (Chemicon AB5855) 1:1000 in block solution.

2)For glutamatergic synapses (put on cells that did not get anti-GABA A R)

mouse anti-PSD-95 (Affinity BioReagents MA1-046) 1:200

rabbit anti-synapsin 1 (Chemicon AB1543) 1:200

these two together in block 50uL/CS

Move CS back to humdified chamber face up and pipette 50uL antibody dilutions on top of cells. Leave at RT for 1 hour or overnight at 4 degrees. If overnight, wrap outside of chamber in parafilm to prevent evaporation.

Wash cells 3x5’ in PBS. You can directly aspirate off the coverslips and pipette about 100uL PBS onto them 1 at a time.

Make dilutions of secondary antibodies. GABAergic: goat anti guinea pig Cy5, goat anti mouse Cy3. Glutamatergic: goat anti-rabbit Cy5, goat anti-mouse Cy3. All Jackson ImmunoResearch Labs at 1:500. Store secondary stocks at –80, working tube at 4 degrees.

Incubate with secondary for 60’ at RT.

Wash 3X 5’ PBS.

Dip coverslips in water to clear salt, then mount cell side down on slides in one drop of Aquamount. Allow to dry in the dark 20’ before viewing.

Store slides in the dark at 4 degrees for up to a year.

Recipes:

10X PBS (1L): in 800mL dissolve

80g NaCL

2g KCl

1.15g dibasic sodium phosphate (ANHYDROUS)

2g monobasic potassium phosphate (ANHYDROUS)

1g CaCl2 (ANHYDROUS)

1g MgCl2 (HEXAHYDRATE)

ddH2O to 1L

filter sterilize and store RT

(cannot autoclave with the Ca and Mg – if necessary, make Ca and Mg free, autoclave, and then add from sterile 1M CaCl2, MgCl2 stocks.

1X PBS

Dilute 100mL 10X PBS into 1L total volume with ddH2O

pH with pH meter to 7.4.

store RT

8% paraformaldehyde

DO EVERYTHING IN THE FUME HOOD, INCLUDING WEIGHING

8g paraformaldehyde

8g sucrose

in 50mL ddH2O

stir and heat at setting “3”

add 20uL 1N NaOH and stir until clear

add 10mL 10X PBS

adjust pH to 7.2 with pH paper NOT machine

adjust volume to 100mL

filter through whatman paper

aliquot in 10mL and store –20 or –80 for 6 months.

Block solution (10mL)

8.3mL 1X PBS

1.6mL Goat serum (Gibco/Invitrogen)

0.1mL 20% Triton-100