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hillaryblack's Content

There have been 4 items by hillaryblack (Search limited from 11-August 19)


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#181806 any suggestions for a good mouse anti-phospho ERK1/2 antibody for immunofluoresc

Posted by hillaryblack on 25 April 2019 - 07:44 AM in Histology and Pathology

Hello,

I am looking for a mouse anti-phospho ERK1/2 to perform immunofluorescence staining on human iPSCs.

Can anyone suggest a good antibody for this purpose? 

 

I have experience with rabbit anti-phospho ERK1/2 #4370 from Cell signalling that works pretty well after methanol permeabilization, but now I need to consider mouse alternatives for double staining.

 

Many thanks in advance,

 

Ilaria




#180698 anti-HA immunofluorescence on HEK 293 cells

Posted by hillaryblack on 27 August 2018 - 11:09 AM in Histology and Pathology

Thank you for the advice. I will try to change the fixation step and see how it goes



#180696 anti-HA immunofluorescence on HEK 293 cells

Posted by hillaryblack on 27 August 2018 - 08:03 AM in Histology and Pathology

Hi,

thank you for your reply.

I seed my cells onto polilysine-coated coverglasses and they adhere quite well.

I already performed immunofluorescence on transfected  HEK293 using other antibodies with no issues.. I'm encountering problems only with these anti-HA.




#180693 anti-HA immunofluorescence on HEK 293 cells

Posted by hillaryblack on 27 August 2018 - 02:12 AM in Histology and Pathology

Hello,

I've been trying to perform immunofluorescence on HEK293 cells overexpressing HA-tagged proteins.

I tried anti HA 12CA5 (Roche) 1:100, anti HA (C29F4) rabbit mAb from Cell Signalling (1:1000 and 1:500) and anti-HA (6E2) mouse mAb from Cell signalling (1:100).

I performed fixation with PFA 4% for 15 min at rt, then I blocked with 5% NGS, Triton 0.3% in PBS for 1 h at RT. The incubation with the primary antibodies was done either overnight at 4 degrees or overnight at rt, followed by  incubation with secondary antibodies (Alexa Fluor conjugated from Invitrogen) for 2 h at rt. I used Triton 0.3% also during the washing steps.

The antibody from Roche doesn't seem to work at all, while the others give a very faint signal.

I also checked the expression of my constructs by Western blot and they are definitely expressed.

Does anyone have experience with these antibodies or any tips to improve the staining?

 

Thank you!

 

 

 





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