Immunocytochemistry showing high background - (Mar/31/2008 )
I am doing suspension culture for limbal stem cells. These cells are not adherent so i am taking them on poly lysin coated slide and then fixed with methanol or acetone....But I am facing problem with ICC...Getting high background whatever antibody i am using its giving totally dense brwon stain even in negative control also....what could be the reason....n what could be the avenues....????
-Shweta Sharma-
QUOTE (Shweta Sharma @ Mar 31 2008, 06:42 PM)
I am doing suspension culture for limbal stem cells. These cells are not adherent so i am taking them on poly lysin coated slide and then fixed with methanol or acetone....But I am facing problem with ICC...Getting high background whatever antibody i am using its giving totally dense brwon stain even in negative control also....what could be the reason....n what could be the avenues....????
What antibodies are you using and at what concentration? What are you using for your negative control?
-Clare-
QUOTE (Clare @ Mar 31 2008, 11:32 PM)
QUOTE (Shweta Sharma @ Mar 31 2008, 06:42 PM)
I am doing suspension culture for limbal stem cells. These cells are not adherent so i am taking them on poly lysin coated slide and then fixed with methanol or acetone....But I am facing problem with ICC...Getting high background whatever antibody i am using its giving totally dense brwon stain even in negative control also....what could be the reason....n what could be the avenues....????
What antibodies are you using and at what concentration? What are you using for your negative control?
I am doing immunocytochemistry for cytokeratin 3, its a surface marker. Iam using 1:50 dilution (Chemicon) of this Ab. Basically as -ve control I am just eliminating the primary Ab from the reaction rest is same.
-Shweta Sharma-
QUOTE (Shweta Sharma @ Apr 3 2008, 06:40 PM)
QUOTE (Clare @ Mar 31 2008, 11:32 PM)
QUOTE (Shweta Sharma @ Mar 31 2008, 06:42 PM)
I am doing suspension culture for limbal stem cells. These cells are not adherent so i am taking them on poly lysin coated slide and then fixed with methanol or acetone....But I am facing problem with ICC...Getting high background whatever antibody i am using its giving totally dense brwon stain even in negative control also....what could be the reason....n what could be the avenues....????
What antibodies are you using and at what concentration? What are you using for your negative control?
I am doing immunocytochemistry for cytokeratin 3, its a surface marker. Iam using 1:50 dilution (Chemicon) of this Ab. Basically as -ve control I am just eliminating the primary Ab from the reaction rest is same.
are you using a secondary antibody?
-Clare-
QUOTE (Clare @ Apr 4 2008, 03:53 AM)
QUOTE (Shweta Sharma @ Apr 3 2008, 06:40 PM)
QUOTE (Clare @ Mar 31 2008, 11:32 PM)
QUOTE (Shweta Sharma @ Mar 31 2008, 06:42 PM)
I am doing suspension culture for limbal stem cells. These cells are not adherent so i am taking them on poly lysin coated slide and then fixed with methanol or acetone....But I am facing problem with ICC...Getting high background whatever antibody i am using its giving totally dense brwon stain even in negative control also....what could be the reason....n what could be the avenues....????
What antibodies are you using and at what concentration? What are you using for your negative control?
I am doing immunocytochemistry for cytokeratin 3, its a surface marker. Iam using 1:50 dilution (Chemicon) of this Ab. Basically as -ve control I am just eliminating the primary Ab from the reaction rest is same.
are you using a secondary antibody?
yes I am using 2ndry Ab provided by kit (Novacastra), its a biotinylated universal Ab
-Shweta Sharma-
what enzyme are you using to visualize the icc? is it hrp? if so, then you may need to pretreat your tissue with peroxide to knock out any endogenous peroxidase.
-mdfenko-