What is the purpose of dehydrating in ethanol and soaking in xylene before mounting the coverslips?

By dehydrating and soaking in xylene, the tissue is properly dehydrated prior to application of permamount. Without this step, the permamount would take longer to dry, and the coverslip would most likely slide during visualization. I've also noticed that visualization of the slides prior to curing of the permamount increases the amount of air bubbles formed when the slide is over the lamp.

purpose of citrate wash

Boiling the sample in the sodium citrate solution allows for heat induced antigen retrival. However, this step is not always necessary, and is highly dependent upon the antigen or epitope you are attempting to stain for.

I have a problem with ABC kit, because it is expired, how many time can i use the kit after it expired.

If i put the unmasking in hot bath and it is in boiling point, the results change? what is the chemistry principle of unmasking use.

And i habe trouble with GFAP because it has nonespecific marking with neurons and don´t mark astrocites i think that is the time of incubation of primary antibody. but what can I do?

Hi Karolina,

I am from Vector Labs, manufacturer of the ABC kit described in the video. My name is Craig Pow and I head the technical service department. We were invited to respond to your inquiries by the management at JoVE to perhaps offer you some insightful information as we are very familiar with the product of course.

You have written essentially three inquiries. From our perspective, none of them are particularly in reference to the video and two of them do not pertain to the Vectastain ABC detection reagents. Regardless, we hope the following information is helpful.

The Vectastain ABC kits are given a guaranteed workable shelf-life of 12 months from the time of receipt. We know the kits will work well without any loss of sensitivity or performance during this time period when stored appropriately at 4 degrees Celcius. It has been our experience, and that of many investigators, that this one year timeframe is by no means a "drop dead" date. In other words, the ABC kits can be used well beyond this one year date, in some cases two to three years, again without any loss of performance. The reagents in the kits are very stable under the refrigerated conditions and this is true for the numerous Vectastain ABC kits we offer that consist of concentrated and Ready-To-Use formats with different enzyme systems. You do not state which Vectastain ABC kit you have or how long the product has been expired, so I do not think we can provide you with further specifics.

The following recently published references should provide you with details and current opinions regarding antigen unmasking in immunohistochemistry:

1) Shi, S.R. et al (2007) J. Histochem. Cytochem. 55(2):105-109

2) Leong, T. Y-M. and Leong, A. S-Y. (2007) Adv. Anat. Pathol. 14(2):129-131

3) D'Amico, F. et al (2009) J. Immunol. Methods 341(1-2):1-18

Your third inquiry appears to be more of a question concerning specificity of the primary antibody rather than a background staining issue. Indeed primary antibodies directed against GFAP (glial fibrillary acidic protein) should bind to astrocytes. From your stated observations the primary antibody is not recognizing the correct cell type in your tissue. I am unclear if you are applying antigen unmasking techniques in this application. Certainly inappropriate antigen unmasking parameters (such as the salt used, pH, temperature, time) may alter tissue properties to generate inaccurate staining. This is where positive and negative controls would be vitally important to validate the staining results on the test sections. Primary antibody incubation times for standard sections cut at < 10 micrometers usually run around 30 to 60 min at room temperature. In some cases overnight incubation at 4 degrees Celcius is required where poor affinity or avidity for the target antigen occurs. We do offer a Troubleshooting Guide in pdf format on our website that describes controls to use in the absence of staining or if background staining is a problem. We hope you find this Guide and the information presented here helpful.

Regards,

Craig Pow