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I need to develop double IHC analysis on formalin-fixed paraffin-embedded (FFPE) tissue sections of human tumors specimens, and after we have to do laser capture microdissection of positive cells. I already have two primary antibodies directed against CD44 (produced in mouse) and against ALDH1 (produced in rabbit). I'm looking for double staining kit (or all of the individual components which analysis needs) with alcohol insoluble chromogens because after IHC and before laser cature microdissection I have to dehydrate the slides. Thank you for your time and consideration.
Asked on Feb 21 2012
Thank you for contacting us.
We do not currently offer double staining IHC kits. Your staining conditions are challenging as, of the two common HRP chromogens, only DAB is alcohol insoluable. While of the common AP chromagens, only BCIP/NBT is insoluable. This means that to do double labeling you will have to do a sequential stain first using one detection system and then with the other.
I have collected a list, with links, of the reagents which you would need for this type of staining.
1) After de-parrafinizing and re-hydrating the samples, perform an antigen retrieval step treating with Cirate buffer pH6.0,https://www.abcam.com/10x-Citrate-Buffer-pH-6-0-ab64214.html, in the microwave or pressure cooker. After performing the antigen retrieval rinse in cold tap water for 10 minutes.
2) Rinse slides in TBST,https://www.abcam.com/20x-TBS-T-with-Tween-20-ab64250.html.
3) Performing a blocking step using protein block,https://www.abcam.com/Protein-Block-ab64226.html.
4) Incubate sections with your first primary antibody, for clarity I will assume the CD44 antibody, raised in mouse.
5) Wash with TBST.
6) Block endogenous peroxidases using aHydrogen Peroxide Blocking Reagent,https://www.abcam.com/Hydrogen-Peroxide-Blocking-Reagent-ab94666.html.
7) Incubate your tissue with a biotinylated secondary antibody, in this instance anti-Mouse IgG,https://www.abcam.com/Biotinylated-Goat-anti-Mouse-IgG-H-L-Ready-to-Use-ab64255.html.
8) Wash with TBST.
9) Detect your first antibody usingStreptavidin Peroxidase (Ready to Use)https://www.abcam.com/Streptavidin-Peroxidase-Ready-to-Use-ab64269.html. Note that this does not containa serum or BSA solution as diluent. Serum may contain biotin therefore competing Streptavidin binding with biotinylated secondary antibody, therefore reducing binding activity.
10) Apply your chromogen, DABsubtrate,https://www.abcam.com/DAB-Substrate-Kit-ab64238.html.
11) Wash with TBST.
12)Performing a second blocking step using protein block,https://www.abcam.com/Protein-Block-ab64226.html.
13)Wash with TBST.
14)Incubate sections with the your second primary antibody, this time ALDH1, raised in rabbit.
15)Wash with TBST.
16)Incubate your tissue with a biotinylated secondary antibody, in this instance anti-Rabbit IgG,https://www.abcam.com/Biotinylated-Goat-Anti-Rabbit-IgG-H-L-Ready-to-use-ab64256.html
17)Wash with TBST.
18)Detect your first antibody usingStreptavidin Alkaline Phosphatase (Ready to Use)https://www.abcam.com/Streptavidin-Alkaline-Phosphatase-Ready-to-Use-ab64268.html.
19) Apply your second chromogenAlkaline Phosphatase chromogen (BCIP/NBT) - Ready to Use,https://www.abcam.com/Alkaline-Phosphatase-chromogen-BCIP-NBT-Ready-to-Use-ab7468.html.
18) Wash with TBST, counterstain if desired.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Answered on Feb 21 2012