I want to start immunostaining at our lab with a very small budget and basic facilities. I have experience of fluorescent immunostaining of acetone fixed cells as well as PFA fixed saponin permeabilized cultured cells. I want to try immunostaining of formalin fixed, paraffin embedded tissue sections for viral antigen localization. Immuno-histochemistry seems more complicated and costly for starters. I need suggestions on followings:
1. Why do people prefer immunohistochemistry over fluorescent immunostaining?
2. Will polyclonal antibodies raised in rabbits using killed commercial viral vaccines serve well as primary antibodies?
3. Since viral antigens are expected to much more numerous than tissue antigens, does one need unmasking?
4. Will FITC serve well for secondary antibodies and how long will the slides remain fluorescent?
5. Is 10% neutral buffered formalin good enough for immunostaining and can the rest of the tissue processing be kept same?
6. Can I use serial sectioning and stain one section with H&e while next one wiith immunostaining for bright-field/fluorescent imaging?
Too many questions, but suggestions will be highly appreciated!
IF is generally preferred over IH for tissue sections because of the high auto-fluorescence that occurs in fixed tissue. This usually interferes with or prevents obtaining high contrast fluorescence images.
As to how long fluorescent staining will persist upon storage, this will vary greatly depending upon your antigen, the particular antibodies that you use and the storage conditions. In general, it is always best to examine and photograph your sections as soon as possible (within hours) after staining. The reason for this is related to the antibody antigen interaction itself. This interaction is electrostatic and therefore has an off rate whereby dissociation is constantly occurring. So, the longer you wait to look at the tissue sections, less specific and more background fluorescence will be present.
Iif you do choose to store the stained sections, store them at -80 C to minimize fluorescence degradation.
ad1. Reading, storing and sharing slides for light microscopy is more comfortable, especially if you have a big amount of slides to read and don't want to sit in the dark the whole day.
ad2 and 3. I doubt, that this kind of antibodies will work. It depends on the denaturation of the antigen during formaldehyde-fixation. And this depends more on the composition of the antigen than on the amount of antigen. So it is a matter of trial. That's the same for the need of antigen-retrieval. But mostly HIER improves the result.
ad4. As a rule of thumb florescent slides should be read in a week, but cold storage and anti-fading mounting media should improve the stability.
ad5. 4% NBF is the standard fixative for FFPET and IHC. The point is the neutral buffering. Processing needs no changes.
ad6. this is the usual method.
Read the basics in http://www.dako.com/at/index/knowledgecenter/kc_publications/kc_publications_edu/immunohistochemical_staining_methods.htm?undefined&from=fb2_ihc_guidebook#.VY5NgU0w_IU
good luck
the answers have mostly been given, just my two cents worth below;
1. As Robert says, if you do IF on FFPE tissue, there will be a lot of background immunofluorescence from the tissue itself. If you are looking to detect virus, this background will surely impede examination. And as Gudrun said, IH slides are much more practical and can be kept essentially forever.I am not sure that IF is that much of a cost saving compared to IH. The major cost will be the primary antibodies, which you have to pay for whether you use one approach or the other.
2. I think what you are asking is whether you can raise your own primary antibodies by immunizing rabbits and then using their serum ? Personally I would discourage you from trying this, it represents a lot more work than you might think, and in the end may not work. The serum must be affinity purified to select for the antibody of interest, and even when you do that you don't know if the antibody will work on your sections. It is much better to buy a good primary, where the supplier has done the purification, testing and quality control.
3. It depends on the circumstances. Detectable virus in tissue might be quite difficult to find in general, so you shouldn't assume that there will be lots of it. Unmasking is generally recommended in FFPE tissue - it may not be required, it depends on the antibody you are using, but you should assume that you will need it.
4 see 1.
5. Yes 10% NBF is the usual fixative. I think what Gudrun meant above was 4% paraformaldehyde, which is the same concentration that you will get in 10% formalin.
6. Yes - not only can you do it, but you should do it. It is important to have an H&E stain as a reference, to know exactly what you are looking at, which isn't always clear with IF or IH stains.
If you still prefer IF, as I do, I would suggest using methanol fixation (70%) which sometimes gives better results than PFA. Next, the secondary antibodies should be labeled with Cy3 (it is more stable, than FITC). It is essential to embed the material in antifade solution. Vectashield is very expensive, so it is better to use glycerol with PPD and Moviol - there is a protocol in the internet. Such slides can be kept at -20 for weeks.
Thank your every one for excellent suggestions! IHC seems essential and fluorescent staining does not seem promising, specially due to fluorescent signal reduction in longer term. Thank you again!
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies