Basic Immunofluorescent (IFA) Staining Protocol
IFA Information: A very basic protocol is linked below for free floating tissue sections. This protocol has been tested and works very well for brain and vestibular (inner ear) tissues. Note that different tissue types may behave differently so modifications may be necessary.
IFA Tips and Tricks
- Check the status of antibodies frequently; particularly fluorescently labeled secondary antibodies have a typical shelf life @ -20F of about 2 yrs with minimal freeze-thaws. Primary antibodies seem to fair better but still have a limited shelf life.
- Always note storage conditions! Store antibodies and reagents at their optimal temperature and add glycerol or other anti-freeze when noted on the spec sheet.
- The freshness of the fixative and the type of fixative can make a huge difference for your results. Check the antibody spec sheets for your antigens of interest. You may need to test out several different fix conditions before getting it right.
- Time, temperature and concentration all matter for all of the steps! Pay attention to what’s working for you and make careful notes as you work so you know where things may differ and their effect on the end result.
- Different lots of antibody (or manufacturers) may perform very differently. It’s always good to test out a small subset of slides before moving forward with a big experiment when using new lots or new antibodies.
- Check the literature! Often tips and tricks for particular antibodies can be found in the methods section of a reference paper. Also the Journal of Comparative Neurology keeps an antibody database on their website.
Mounting Medias For Chromogenic Studies
- The type of mounting media used to permanently preserve the staining and tissue is important. There are a number of permanent mounting medias available - below is the preferred for DAB and related studies. If you anticipate examining (or counting) the slides using oil (100x), a low viscosity mounting media is recommended.
Recommended Permanent Mounting Media (Chromogenic Studies Only, Not For Fluorescence Studies)
- Cytoseal 60 (Low Viscosity)
- Richard-Allan Scientific (available through VWR) cat # 8310-16 (no commercial interests)
Mounting Medias for Fluorescence Microscopy
- The most important issue is to make sure the anti-fade in the mounting media is stable and that the mounting media does not ‘auto fluoresce’. All anti-fade reagents have a shelf-life – so need to check the expiration dates. Recommend using for no more than 12 months when stored properly.
Recommended Fluorescent Mounting Media
- Mowiol (see recipe and information below) which is particularly good for studies examining eGFP expression. This is an aqueous mounting media – so the slides can be held in the refrigerator for a period of time but do not freeze. Cost effective method of maintaining fresh anti-fade. Mowiol reference document
- Prolong Gold is a permanent mounting media for immunofluorescence. Available from Invitrogen – (no commercial interests). Requires short cure time.
- Slo-Fade is a permanent mounting media for immunofluoresnce. Available from Invitrogen – (no commercial interest). Minimal cure time so can view slides almost immediately after mounting.
**Note that some of the mountants may come with nuclear counterstain DAPI added into the mounting media. Our experience is that DAPI stains much more evenly when used as an additional staining step during the staining protocol as opposed to adding it in the mountant. If you already have mountant with DAPI, you can still add the additional DAPI staining step to achieve brighter, more uniform DAPI stained nuclei.
Laser Capture Microdissection Microscopy