This may seem like a simple question, but I have never done any form of histology. I have rats with adjuvant-induced arthritis in the left knee joint, and I wish to see the fluorescence from a labeled macromolecule to the site of inflammation, as well as stain the peripheral nerves with proper antibodies for IHC. I am doing both frozen section and paraffin-embedding. Any suggestions, ideas, or advice, on how to section the joints and perhaps if this is doable? I wish to see the fluorescence from the nerve stains and from the macromolecule together.
precisely what do you want? according to your question 1: histology Technique;2:IHC method;3:sectioning technique of joints or nerves?
Niseel body staining and Ag nitrate staining for nerves is suitable
After your experiental treatemnt/exposure (whatever) , rat knee joints should be removed, trimmed, fixed in 4% paraformaldehyde and then decalcified in 10% EDTA for 2 weeks. The tissues should be embedded in paraffin and sliced (5 μm) for the histological examination and immunohistochemical analysis both in longitudinal and transverse directions.Paraffin sections of knee joints should be firstly stained with hematoxylin and eosin (HE) and evaluated by two trained observers unknown the sample source. If significant difference are identified between HE control and treated samples - it is good, if not - the difference might be seen later during specific antibody staining.
TheIHC procedures (immunohistochemistry) should be performed with classical protocols available online. Briefly, tissue sections should be hydrated, rinsed and microwave-treated in citrate-buffered saline. Sections can be than treated with 3% hydrogen peroxide followed by incubated in 5% goat serum ( rabbit or mouse serum it depend on your antibody). Subsequently, sections should be incubated overnight at 4°C with primary antibodies (or just follow the protocol given to you by the antibody manufacturer). I have also ttached a paper with some protocols. Cheers!
Paraffin embedded section for S-100 protein stain would suffice for nerve IHC. S-100 stains the schwann cells of nerves and a whole lot of other cells. So please check that stained area corresponds to nerve tissue (you may require a H&E section of the same for this). There is no fluorescence observed on S-100 IHC. So your "wish to see the fluorescence from the nerve stains and from the macromolecule together" may not be feasible.
The protocol can be found here https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152214/
and good protocol is here
http://www.protocolsonline.com/histology/sample-preparation/paraffin-processing-of-tissue/
Immunohistochemistry:
Yes, S100. Also:
Neuroendocrine markers: CD56, chromogranin, synaptophysin, neuron specific enolase (NSE)
Neuroglial markers: neurofilament (NF), glial fibrillary acidic protein (GFAP), NeuN, (as well as CD56, chromo/synapto)
A general marker of nerves is AcT(Acetylated tubulin) antibody you may use by means confocal immunoflurescence and PFA % fixed sections
For studies on human cruciate ligaments many years ago, we fixed in formalin, embedded in paraffin, and stained sections with silver methods and (separately of course) with immunohistochemical methods for neurofilament protein. I don't recommend doing an S100 immunostain as it will miss small nerve fibers and may mark cells that are not nerve-related (not everything you will see with S100 is a Schwann cell, indeed chondrocytes are S100 immunopositive as are many histiocytes. You will need to decalcify your tissues if you are including the actual bone tissue, in the sections (we didn't need to do that with ligaments that had been removed from the bone attachments.)
Thank you for all the answers. I guess I was just looking for general advice, if anyone has tried to do IHC and nerve staining on rat knee joints and how was his or her experience.
Dear Laura
From what I saw you will do your histological study in paraffin, in this way, it will be necessary a descalcification of the samples, followed by inclusion in paraffin and cuts with approximately 4 micrometers, following the processing for immunohistochemistry. I describe NF-Kb, but it can be used for other antibodies, if this technique is well documented, you may need some help in standardizing the methodology.
Follow technique
Technique - streptavidin-biotin. Cuts made with approximately 4 mm thick were fixed on glass slides previously prepared with 20% 3-aminopropyltriethoxy-silane (Sigma Chemical, St. Louis, USA). For antigen recovery, the slides were dipped in citric acid solution for 40 minutes, at a temperature between 95 ° C and 98 ° C. The slides were incubated in polyclonal anti-NF-κB antibody (Rabbit anti-human mouse and goat, NFKappaB Lab. Zimed, 1: 200). Samples were subsequently incubated in secondary antibody and tertiary complex (LSAB + Peroxidase, Dako Corporation, Carpinteria, USA). Reaction development was with 0.025% diaminobenzidine (DAB) solution (3,3 'diaminobenzidine, Sigma Chemical, St. Louis, USA) for ten minutes. The slides were counterstained with Mayer's hematoxylin, dehydrated, diaphanized and mounted with Permount® (Fisher Scientific, Fair Lawn, USA). Table VI demonstrates dehydration and diaphanization:
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Dear Laura
I think suggestion suggested by Marcos is appreciable. you should go for that
I think that NF antibody is a suitable maker used for both central and peripheral nervous system.It is simply an antibody to a type of intermediate filament that serves as major element of the cytoskeleton supporting the axon cytoplasm, no a neuronal marker at a greater extent .
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