'ChAT' is the abbreviation for Choline Acetyltransferase. As the name suggests, this is an enzyme ('-ase') that transfers an acetyl group onto the choline molecule. That is, it converts choline into acetylcholine. Neurons that contain ChAT are putative cholinergic neurons.
In the Newman Lab, we study these neurons from several directions. In some experiments we lesion these cells to see what the consequence is for the health of the rest of the brain. In which case, we stain for ChAT to see how thorough our lesion was. In other experiments, we express an opsin that allows us to activate or silence the cholinergic neurons to test the impact on animal behavior or brain function. In which case, we stain for ChAT to see if our opsin was expressed exclusively in ChAT neurons and to determine what percentage of the ChAT neurons we successfully transfected. In other studies, we study how the health of ChAT neurons
Wash with pure PBS 5 min x 2 times
2L PBS wash
0.5% Triton (10 ml)
5min x 4 times
Put the marker on the slides to form wells around each slice
Donkey serum in PBS 10% 20 or 30 min
50 μl per slide / for floating tissue 6-8 slices per well in 1-2 ml No drying of the slides
Tap off serum
Primary antibody
Goat MAB305 Anti-Choline Acetyltransferase Antibody, clone 1E6 (Milipore-Chemicon) or Mouse MAB305 Anti-Choline Acetyltransferase Antibody, clone 1E6 (Milipore-Chemicon) 1/100 or 1/200
50 μl per slide
Incubate overnight at 4°C in constant agitation
The primary antibody is prepared in pure PBS or in 1-5% goat serum PBS
PBS Wash 5min x 4 times
Secondary antibody
Donkey anti-goat IgG(H+L) secondary Antibody Alexa Fluor 594 (ThermoFisher) or Donkey anti-mouse IgG(H+L) secondary Antibody Alexa Fluor 594 (ThermoFisher) 1/200 or 1/350
50 μl per slide
60-90 min at room temperature
The secondary antibody is prepared in pure PBS or in 1-5% goat serum PBS
PBS wash
5 min x 4 times
Nuclear labeling with DAPI (optional)
Mount slides with Vectashield mounting medium and sealing the edges of the coverslip with clear fingernail polish.
Description/Samples
Primary:
https://www.emdmillipore.com/US/en/product/Anti-Choline-Acetyltransferase-Antibody,MM_NF-AB144P
Secondary:
1ºAntibody Staining
Carefully move sections from cryoprotection to PBS in petri dish using transfer pipette
Wash the sections with PBS-T (PBS+0.1%Tween-20) -- 3x5 minutes with agitation
Permeabilize the sections in 0.4% triton X-100--- 20min with agitation
Block in 10% NDS (normal donkey serum) in 1% BSA for 1 hour @ R.T with agitation
Incubate in 1ºAntibody (Dilution 1:500) in 0.5% BSA @ 4ºC for overnight
_____ul of primary antibody in .5% BSA
Use tubes to incubate primary antibody (overnight @4ºC)
2ºAntibody Staining (all done on floating sections)
Wash in PBS-T 3x 5minutes with agitation
o Incubate in 2ºAntibody ( Dilution 1:500) + 0.5%BSA for 60min in dark
______ul of secondary antibody in .5% BSA
Rinse in PBS-T 3x5 minutes
Incubate in DAPI (1 drop/2mLs PBS) for 5 minutes
Wash 2x5 minutes in PBS-T
Wash 1x5 minutes in PBS
Float sections in Tris
Carefully mount the sections
Add ~130 ul antifade mounting media and coverslip
NOTES
Immunofluorescent Protocol-Coplin jars
1ºAntibody Staining
Carefully move sections from cryoprotection to PBS in petri dish using transfer pipette
Wash the sections with PBS-T (PBS+0.1%Tween-20) -- 3x5 minutes with agitation
Use mesh inserts in 12-well plates- 1-2 sections per well
Permeablize the sections in 0.4% triton X-100--- 20min with agitation
Use mesh inserts in 12-well plates- 1-2 sections per well
Float sections in water
Carefully mount the sections onto Superfrost Plus slides using transfer pipette and paintbrush- look under scope in 1131c to make sure there are no folds or wrinkles in tissue
Put slides in the desiccator to dry for several hours
Remove lid of desiccator and insert slides
Close lid
Turn vacuum (Yellow knob) to the right to turn on vacuum. You should hear air being sucked out. Allow to run for 3 minutes.
Turn vacuum off (turn knob left) and clamp the tube shut using black clip to stop air from leaking in
Let sit for several hours
Using the pap pen, draw a square around sections and allow to dry.
Dip into PBST in Coplin jar for 1 minute
Add wet paper towels to humidity chambers
Block in 10% NDS (normal donkey serum) in 1% BSA for 1 hour @ R.T in humidity chambers- you will only need around 150ul per slide
To make 5mls of 10% NDS (calculate volume you will need based on number of slides and sections
500uls of NDS in 4.5mls of 1%BSA
Incubate in 1ºAntibody (Dilution 1:500) in 0.5% BSA @ 4ºC for overnight in humidity chambers
To make 5 mls primary antibody
2.5 mls 1% BSA
2.5 mls PBS
_____ul of primary antibody
Use humidity chamber to incubate primary antibody (overnight @4ºC)
2ºAntibody Staining
Wash in PBS-T 3x 5minutes with agitation in coplin jar
Incubate in 2ºAntibody ( Dilution 1:500) in PBST for 90min @RT
In humidity chamber
All steps from this point forward are in the dark or foil
Rinse in PBS-T 3x5minutes with agitation in Coplin Jars
Incubate in DAPI (1 drop/2mLs PBS) for 5 minutes on slides
Wash 3x5 minutes in PBS-T in Coplin jar
Wash 1x5 minutes in ddH20 in Coplin jar
Coverslip
Add ~170 ul antifade mounting media and coverslip
Dry slides in drawer for at least 1hr, but you can let them dry overnight
Store dried slides in fridge