Boster Bio Life Science Blog

Why Optimize Antigen Retrieval in IHC?

Optimizing antigen retrieval in immunohistochemistry (IHC) is critical for unmasking epitopes that become concealed during formalin fixation and paraffin embedding. This step enhances the binding efficiency of primary...

Overview of Antigen Retrieval Techniques

Antigen retrieval is essential in immunohistochemistry (IHC) due to protein cross-linking caused by formalin fixation, which masks antigenic sites and hinders antibody binding. Formaldehyde fixation usually generates methylene bridges which cross-link proteins and therefore mask the epitope of interest. It is essential to unmask the antibody...

The purpose of using a secondary antibody is to amplify the primary antibody and antigen interactions. Before choosing a secondary antibody for your experiment, ask yourself the following 3 questions to help you choose the appropriate one:

• What is the host species of the primary antibody?

  The secondary antibody should be raised against the host in which the primary antibody was raised. For example, if the primary antibody was raised in rabbit, you will need an anti-rabbit secondary antibody raised in a species other than rabbit.

• What is the isotype of the primary antibody?

  The secondary antibody has to be directed specifically against the isotype of the primary antibody. For example, the polyclonal primary antibodies are generally of the IgG isotype and thus the corresponding (species specific) secondary antibody needs be an anti-IgG antibody. The monoclonal

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Counting cells before the staining procedure and the analysis/live sort is not up for debate! Cell numbers affect the staining quality, the FACS instrument reading, as well as the efficacy of any downstream assay, in case the cells are live sorted. Furthermore, cells are steadily lost during the staining procedure and the recovery of most sorters is a little above 50% − it is therefore imperative that enough cells are available at the start of the experiment to compensate for the inevitable losses. There are various methods available for counting cells:

Hemocytometer

Hemocytometers are manual counting chambers and were first developed for blood cell counts. It consists of a thick glass slide onto which a gridded chamber is affixed. The four corner squares of the grid measure 1 mm x 1 mm and each square is further subdivided into 0.05 mm x 0.05 mm squares. A glass coverslip, made to specifications of the hemocytometer, can be placed 0.1 mm above the marked grid, thereby creating a space of known volume.

The cells are counted using a 4x or 10x objective lens over an area of 1 sq mm and the counting is repeated for a total of four such areas and the result is averaged. The formula for counting cells for each area is (average number of cells * 10 ⁴ * dilution factor). To count the viable cells, use Trypan Blue exclusion method. Save some time and trouble with Boster’s ready-to-use Trypan Blue Assay Kit AR1175, see the kit below!.

Although the easiest method and least expensive, it is also labor intensive and allows for inaccuracies of the human eye. The image below depicts a typical hemocytometer counting grid.

Automated Cell Counters

They perform on the same principle as the hemocytometers but with the added advantages of speed and precision. Automated cell counter models have been developed by man...

With great versatility (in FACS panels and protocols), comes great responsibility in choosing the right controls. Take a quick look to make sure you have included all the appropriate ones! Here’s a convenient mini guide to FACS staining controls:

Are you preparing for a FACS experiment? Here’s a 10 point checklist to help you prepare for your FACS sorting experiment:

• Perform a cell viability count using the trypan blue exclusion method.
• Remove any existing clumps in the cell suspension by sieving cells through a nylon mesh.
• Adjust the cell concentration to 107-108 per ml.
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