I check the location of my protein in Leishmania cell. I obtain positive results, the DAPI stain is ok, but I obtain I a high level of non-specific binding, probably due to hydrophobic interaction. I use this for blocking BSA 4% in PBS (1X), 1h at RT. I incubated my primary antibody (polyclonal) over night at 4ºC and the secondary (Donkey anti-rabbit IgGs-conjugated with Alexa 488) at 1:500. I am checking whether any people used goat serum for blocking. Does anyone have any suggestions?.
you could try goat serum - however usually you should block with the serum of the species the secondary antibody was raised in. you could try different washing buffers (are you using tween in the PBS?) higher dilution of primary and secondary antibody, different fixation protocol.. . how do the controls look like?
I fix my parasites in 1% methanol-free formaldehyde, block in 0.1% (v/v) Triton X-100, 0.1% (w/v) BSA, in PBS, and perform all my antibody incubations in this blocking buffer. I also filter the solutions prior to use.
Harvest promastigotes 1000 g 5 min then wash twice in PBS
Fix in 1% methanol-free formaldehyde (Thermo Scientific) for 10 min
Permeabilse in 0.1% Triton X-100 for 10 min
Add 0.1 M glycine for 10 min
Centrifuge 2000 rpm 10 min
Resuspend in appropriate volume PBS and add 200 µl cells per well of an 8-well chamber slide (Lab-Tek) then leave to adhere for 10 min
Remove excess PBS
Block in 0.1% (v/v) Triton X-100, 0.1% (w/v) BSA, in PBS for 1 hr
Incubate primary antibody in blocking buffer for 2 h at RT or 4 ºC overnight
Wash wells three times with PBS for 5 min
Incubate secondary antibody (in my case, donkey anti-sheep Alexa Fluor 488-conjugated, used at 1:2000) in blocking buffer for 1 h in a dark box at RT
Wash wells three times with PBS for 5 min
Remove chamber and use VectaShield Mounting Medium with DAPI
Hope this helps.
Dear Amy,
Harvest promastigotes 1000 g 5 min then wash twice in PBS
Fix in 1% methanol-free formaldehyde (Thermo Scientific) for 10 min
you centrifuge?
Permeabilse in 0.1% Triton X-100 for 10 min
Triton in PBS?
you centrifuge?
Add 0.1 M glycine for 10 min
Glycine in PBS?.
Centrifuge 2000 rpm 10 min
Resuspend in appropriate volume PBS and add 200 µl cells per well of an 8-well chamber slide (Lab-Tek) then leave to adhere for 10 min
Remove excess PBS
Block in 0.1% (v/v) Triton X-100, 0.1% (w/v) BSA, in PBS for 1 hr
you check use donkey serum?
Incubate primary antibody in blocking buffer for 2 h at RT or 4 ºC overnight
Wash wells three times with PBS for 5 min
Incubate secondary antibody (in my case, donkey anti-sheep Alexa Fluor 488-conjugated, used at 1:2000) in blocking buffer for 1 h in a dark box at RT
Wash wells three times with PBS for 5 min
Remove chamber and use VectaShield Mounting Medium with DAPI
Thanks for all.
César.
The Triton and glycine solutions are made up in PBS. I do not centrifuge/wash in between the formaldehyde and glycine steps. I perform all of these steps in an eppendorf and only centrifuge after the glycine step. I have always used BSA, either from Sigma, or those molecular biology grade aliquots supplied with NEB restriction enzymes.
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