Better IHC Step 1: Antigen Retrieval
Frustrated by fuzzy IHC staining? Watch this series of short videos to learn how to get better staining and consistent immunohistochemistry results using an optimized protocol.
👉Download the Guide to Successful Immunohistochemistry: https://cst-science.com/mqrpjz
View the Better IHC Playlist: • 4 Steps to Better Immunohistochemistry
Step 1: Antigen Retrieval (this video)
Step 2: Primary Antibody Diluent: • Better IHC Step 2: Antibody Dilution
Step 3: Detection Reagents: • Better IHC Step 3: Detection
Step 4: Chromogen: • Better IHC Step 4: Chromogen
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Transcript: It's Friday night, and you're staring into a microscope, hoping to see positive results from your immunohistochemistry experiment. Unfortunately, all you see is some indistinct fuzziness.
So the data is inconclusive. It's a failure.
We've all been there. Next week you'll come back and work on fixing the protocol.
But where to start?
You probably know that a highly specific, high affinity primary antibody is key to a successful IHC. But did you know that the companion reagents such as buffers, which establish the pH and ionic strength of the system, are just as important? These reagents can influence the binding of the primary antibody to its epitope and dramatically affect the outcome of the assay.
To help you pick the best reagents for your assay and make sure those Friday nights in the lab are worth the effort, we will spend the next several videos reviewing how companion reagents affect IHC, and, as an example, we will describe our experience, optimizing the protocol for one of our antibodies.
Let's get started.
Step One. Antigen Retrieval.
Several methods exist for revealing epitopes that have been massed by cross-linking fixatives like formalin. These include Proteolytic-Induced Antigen Retrieval, which relies on an enzyme like Proteinase K, or Heat-Induced Epitope Retrieval, which uses heat to break apart cross-linked bonds and unwind proteins.
Either method can unmask epitopes, rendering them accessible to the primary antibody and amenable to staining by IHC. At Cell Signaling Technology, we most often use Heat-Induced Epitope Retrieval when testing our antibodies. So this is the method we will discuss in detail.
Heat-Induced Epitope Retrieval, also known as antigen unmasking, involves heating and cooling the tissue sections while they are immersed in a solution with a defined buffering capacity. The pH of the buffer helps keep the proteins unwound after the temperature is returned to normal. So the pH range of the system should be optimized to the antibody-epitope interaction of interest. The slightly acidic buffer citrate is effective at unmasking a wide range of epitopes, but some may require a more basic buffer, like EDTA. We use citrate buffer for Heat-Induced Epitope Retrieval while optimizing the PLK Rabbit mAb protocol because it works for the widest range of epitopes. We kept this method constant as we did a stepwise change of the other companion reagents.
Coming up next, Step Two.
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#antibody #immunohistochemistry #BetterIHC
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