As anyone who performs immunohistochemistry and immunocytochemistry knows, primary antibody supplies can diminish quickly, especially those that are used at high concentrations (such as 1:10, 1:25, 1:50, or 1:100). And with the cost of primary antibodies as high as they are, it can get quite costly to perform these procedures.
In our lab, we perform both immunohistochemistry and immunocytochemistry on cerebellum and hippocampus tissues as well as on glial cell cultures. Our tissues and our cultures are placed onto 13mm diameter glass coverslips which we place into 24-well plates to perform our immunostaining protocols. It has been our experience that in order to get effective and thorough coverage of our antibodies on our tissues and to prevent evaporation, we need to use approximately 250ul of antibody solution per sample (well) in the 24-well plate.
We have found Research Product International's Coverwell Incubation Chambers to be very effective in reducing the amount of primary antibody solution needed to cover our samples. They are available in different size, but we have used the 50ul (13mm diameter) chambers. We use one chamber (coverslip) per sample and only need 50-100ul of antibody solution per sample instead of 250ul per sample. This can significantly decrease the amount of primary antibody needed, which can reduce expenditures a great deal.
It is suggested to press a microscope slide onto the coverwell chamber to form a seal around the specimen to hold reagents in place and to prevent evaporation and sample loss. Although we do not do this in our lab, it would not hurt to do so. We generally do not worry about sample loss since our samples adhere to the glass coverslips. Although we use the 50ul Coverwell Incubation Chambers in our lab, we generally do not worry about evaporation because we use 75ul (instead of 50ul) to cover our tissues and have never experienced evaporation loss.
I would recommend using no less than 50ul and no more than 100ul in the 50ul Coverwell Incubation Chambers. With only 50ul in the chamber, it is possible to get incomplete sample coverage and evaporation results more easily. With more than 100ul in the chamber, it is possible to get spillage and possibly contaminate other samples. We generally place our 75ul of antibody solution in the bottom of the well Coverwell Incubation Chamber, making sure it spreads out evenly and completely coats the entire bottom of the well, then we place our coverslip with our tissue down into the antibody solution. This ensures that the sample is completely saturated in the primary antibody solution.
Research Product International's Coverwell Incubation Chambers are made of a silicone gasket bonded to a hydrophobic plastic. They are microwavable and autoclavable. They can be reused over and over again, although I would recommend only reusing the chambers for the same antibodies to prevent any possible cross contamination. The Coverwell Incubation Chambers are designed for immunocytochemistry, among other uses. In our lab, we prefer to use the chambers only for immunohistochemistry and not for immunocytochemistry simply because it is easier to see a tissue on a glass coverslip than it is to see cultures.
We have also found the Coverwell Incubation Chambers to be an essential tool to perform our TUNEL procedures. The chambers make it easy to keep negative controls and positive controls separated away from each other and away from our test samples. It is an efficient way to keep the various reactants from getting spilled onto the wrong samples.
Research Product International's Coverwell Incubation Chambers are also cost effective. At $116.50 for 50 chambers, it seems a little expensive for something made of plastic and silicone. But, for chambers that can be used over and over, in order to use 75ul per sample instead of 250ul per sample, the cost of the chambers is money well spent. It saves a tremendous amount of money that would otherwise be spent on primary antibodies. I would most definitely recommend Research Product International's Coverwell Incubation Chambers to any lab interested in saving money.
VelvetLee Finckbone, MS
Lab Technician
Strahlendorf Lab
Dept. of Physiology
Texas Tech University Health Science Center