In plants in order to get antibodies to work, you should do the entire process you describe (paraffin, alcohol, xylene...). This process leaves only a dehydrated "skeleton" of the cell (without any solubles) and the proteins "exposed" for the antibody. I imagine that it works in a similar way in rat cells.

So, do you mean that paraffin-embedded sections will stain better than floating ones?

Definitely

Ok, thank you)

From the experimental design shown above it appears that you did not run a positive control with your evaluation of formalin fixed rat brain floating sections. A positive control is one in which all experimental parameters are the same including a known positive within each experiment. A floating section of fresh rat myocardial infarct fixed and processed in an identical fashion and at the same time as your fresh brain tissue should have been included in your experiment. Formalin preserves tissues by binding to free amino groups thus inactivating many enzymes, killing microbes and reducing the rate of tissue decomposition. It does not cross link proteins preventing them from moving out of the tissue. I suspect the procedure you followed resulted in iNOS diffusion from the floating tissue section method you employed. The myocardial infarct control would confirm or refute this possibility. To prevent protein loss from cells in wet mounts glutaraldehyde is the preferred fixative.

iNOS is an inducible enzyme in brain injury.

Thank you for this wonderful idea. I will try to manipulate with fixation and tissue processing in order to check if my troubles were determined by antigen diffusion. An additional problem I see here is the difficulty of obtaining the floating sections of myocardial infarction of good quality, since tissue destruction is very serious there and I am affraid I will lose the area of infarction during the slicing on a vibratome.

Than may be a problem but the margins of the infarct and myocardium will be positive. With your control your goal is to show the method works not necessarily view and photograph good histopathology.

Also not that other known positive tissues, more stable to vibrato me sectioning, may be used in place of myocardial infarction. The purpose of this positive control is to show that the method works.

Another point not completely clear for me is formalin fixation chemistry. You wrote that formalin does not cross link proteins, but doesn't it react with side groups of peptides forming so-called methylene bridges? This opinion is wery widespread; do you think it is wrong?

Every histotechnic-literature tells formaldehyde is a crosslinking fixative. e.g. http://publish.uwo.ca/~jkiernan/formglut.htm

There has been a big amount of research to show the reactions of formaldehyde with proteins (Fraenkel-Conrat, Fox, French and Edsall, ..)

It may be possible, that small proteins in solution are lost anyway.

One should consider the reversibility of formaldehyde - amino interaction when a reducing agent such as sodium borohydride is not employed to stabilise the Schiff's base. With formalin fixed floating sections processed over the course of hours proteins move. The reaction is reversible without reduction. I histology methods formalin fixed tissues are moved to dehydrating solutions then paraffin relatively quickly. They are not removed from the formaldehyde fixative and stored in water or buffer. This is true for all aldehyde fixatives however the dialdehyde, gluteraldehyde, forms a more stable bond with amino groups and holds proteins together better than a mono aldehyde like formaldehyde. Free aldehyde linkages can be blocked by the addition of 10 - 50 mM glycine to the reaction buffer of choice to eliminate background staining.

I may be misinterpreting your protocol. Are you preparing snap frozen formalin fixed rat brain tissue or rat brain tissue well fixed with formalin and cut into sections. Taken from reference given above.

"To be useful as a fixative, a solution must contain monomeric formaldehyde (or methylene hydrate, to be pedantic) as its major solute. Dilution with water breaks up the small polymers in formalin. This process is said to take a couple of days if plain water is used, but to be almost instantaneous when formalin is diluted with a buffer solution at physiological pH (Pearse, 1980). Hydrolysis of the polymers is catalyzed by the hydroxide ions present in the slightly alkaline solution" and "The aldehyde group can combine with nitrogen and some other atoms of proteins, or with two such atoms if they are very close together, forming a cross-link -CH2- called a methylene bridge. Studies of the chemistry of tanning indicate that the most frequent type of cross-link formed by formaldehyde in collagen is between the nitrogen atom at the end of the side-chain of lysine and the nitrogen atom of a peptide linkage (Fig. 2), and the number of such cross-links increases with time (Gustavson, 1956). The tanning of collagen to make leather is comparable to the hardening of a tissue by a fixative (Hopwood, 1969). The fixative action of formaldehyde is probably due entirely to its reactions with proteins. Initial binding of formaldehyde to protein is largely completed in 24 hours (Helander, 1994) but the formation of methylene bridges proceeds much more slowly. Substances such as carbohydrates, lipids and nucleic acids are trapped in a matrix of insolubilized and cross-linked protein molecules but are not chemically changed by formaldehyde unless fixation is prolonged for several weeks."

For immunohistochemistry I have found gluteraldehyde to be superior to formaldehyde. Try both, you need reliable results not a discussion on what fixative do. Brain injury results in an increase in iNOS. Your current method fails to demonstrate this and your experimental design did not contain a positive control.

It's definitely possible that the antibodies will work differently in paraffin-embedded tissue and PFA-fixed frozen tissue. You could also try antigen retrieval on your floating sections, it's not too hard and I've found it has about a 50% success rate. It basically breaks down some of the PFA crosslinks and allows for better antigen exposure. There are a zillion antigen retrieval protocols online, it's basically a 20-min wash in a hot (like 90C) citrate buffer. Good luck! And I agree, add a positive control!

Thank you, Maryann. Actually, in my case HIER had no effect in brain. I tried pH=3, 6, and 9 (two citrate buffers and tris-EDTA, respectively) at 95C for 15 min. On the other hand, paraffin embedded heart tissue stained well even without any HIER.

I did not use frozen tissue; in both cases fixation was perfusion by 4% formaldehyde followed by postfixation in the same solution. The only difference was that hearts with infarction were dehydrated and embedded into paraffin, whereas brains were cut on a vibratome.

yes, definitely.

I use antibody that works perfectly on fresh frozen tissue, and horrible on free floating 4%PFA fixed tissue when I tried incubating the fixed tissue with cold acetone for 7 min instead of permeabilizing the tissues it worked perfectly.

so you might try that as well, good luck.