Hallo Anthony, there are a lot of possibiliteis. Starting with the fixation. Kidneys have a very strong capsula which makes it difficult for the fixative to pass through. I recommand to devide the kidney befor drop in to the formaldehyd.

Also the quality of the paraffin embedding could play a role. Sectioning is the third problem . The reason for this so called weird lines could be : speed of the sectioning , in this case to fast; a non optimal knife angle.

Staining protocol: Deparaffinization of the sections could be unsufficient. That would explain the spotty results. I recommand longer times in Xylol and alcohol (10 min each; solution should be prepared feshly) . You can include the peroxidase inactivation in the deparaffinization (after 2 x Xylol and the 1st ETOH 100% put the slides for 20 min in 100% ETOH + 0,3% H2O2 at 4°C. Then continue rehydration in the following alcohols at room temperature. Antigene retrieval: you can play the time or running different time cycles, like 4 x 5 min.

Dilution media (DM) for all antibody steps: 0.2% Triton in PBS or TBS; Blocking instead of comercial blocking kits ,10% normal Serum (depends on the host of the secondary ab) in DM. Add 1 % normal Serum in the DM of the primary and secondary ab. DAB-Reaction: check the quality of the DAB-soluton: if it is to old precipitates could be decontaminate the solution --> filter it though a 0,2µm syringe filter befor use. Best is to use fresh ones. Use positive controls to check the intensity and quality of the antibody. Hope this recommandation will help you. Good luck!

Hi Anthony,

I must admit that I also had a problem with the IHC analysis. The problem turned out to be DAB (it did not dissolve completely, actually it did not dissolve at all, I had the compound to weigh on the scale, now I use DAB tablets and it's okay). Second problem, biggest ... to IHC when using a secondary antibody with HRP, the picture was poor. Everything changed after application ABC complex (Avidin-Biotin Complex) with a biotin-conjugated secondary antibody. The picture is clear, you can see the expression clearly.

I can send my manual to IHC, maybe there will be a point that is different and maybe help.

Ute Neubacher

Thank you for your input!

1. Problems stemming from poor formaldehyde infusion of the tissue were a leading concern I had. These are Rat kidneys, which typically are ~8.5-17.5 mm wide, so they are pretty dense. We usually cut them in half, but that still only reduces the width to around ~4-8mm. Formaldehyde infusion is suggested to work best with less than ~3mm of tissue width, so it could be that the tissues are too thick for timely formaldehyde infusion delaying the fixation of the tissue-core and leading to differential staining. We do not notice similar problems with the mouse kidneys- which are notably smaller, further supporting the fixative infusion concerns.

2. Unfortunately I do not have any information at this time about how the sectioning was done. However, if there are any fixation problems in the tissue then it is likely that they compounded any artifacts due to slicing. While the concept of "over-fixing" is argued to a myth, many people still recognize that longer fixation times change how the tissue is cut and processed- and these tissues were fixed for an extensive period of time.

3. I will definitely increase the deparaffinization wash times to see if that has an effect. I feel confident about the deparaffinization step performed, but I will definitely try increasing the times in case that plays a factor!

4. The DAB used was a new batch ordered from Cell Signaling Technologies, so it shouldn't have been a problem. That being said, it never hurts being careful, and I'll filter it before use next time!

Thank you so much for your advice!

To solve the fixation problem I recommand to perfuse the animals . I put an image of a perfused rat kidney in the attachment. 6 µm paraffin sections were HE stained.

Izabela Szpregiel

Thank you for your input!

1. The quality of the DAB we have used in the best was... questionable, to say the least- however this run was done with fresh DAB from Cell Signaling that was already in solution (just needed to "add A to B"). Still, like mentioned to Ute Neubacher above- it would be good to filter the DAB before using it.

2. We had a problem with HRP image quality for awhile! We then swapped from a standard HRP secondary to Cell Signaling's SignalStain Boost IHC Detection Reagent HRP Antibodies and we then started getting good signal. Point being, regular HRP-conjugated secondary antibodies left much to be desired.

3. I would love to see your IHC manual if you would not mind sharing it! I think it would also be really useful for anyone viewing this thread that may have similar inconsistencies.

Thanks!

Ute Neubacher -Post #2

Thank you for the beautiful image! We are moving forward with a PFA perfusion to test to test a few things:

1. To see if the blotchy patterns were due to inconsistent fixation (remember, we left our cut-in-half kidneys in formalin jars for 5-8 days before processing and it is likely that the side of the kidney that sunk to the bottom did not have good surface contact with the formaldehyde since it was face-to-face with the surface of the container). I have done perfusions in the past, and they seemed to come out good; I am eager to see any changes in the IHC!

2. To see if more consistent fixation allows for more consistent cutting and tissue processing. Some sources suggest that uneven and/or extended fixation could make it more difficult to cut and section the tissue. I am hopeful that this may repair the "line" artifacts- if they were indeed caused by inconsistent cutting).

3. Something I was not initially aware of- but we have noticed a lot of those "black specks" present in our IHC slides. I was under the assumption it was contamination introduced by some end-stage reagents, but despite using fresher, unopened, and 'histological grade' xylene, ethanol, and mounting solution I continued having such specks. According to an article I read by Leica those black specks that you can see covering the slide in Image 2 could be due to old, "decomposed" unbuffered Formalin that has oxidized into formic acid. Our formalin we use is neutral buffered formalin, but I do have significant doubts about its age. Thus, in our perfusion we will be using fresh PFA, and we shall see if that accounts for the black-speck artifacts we see covering the slide.

Thank you for your input!