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I use methanol fixation (@ -20⁰C for 10 minutes) when performing immunofluorescence assays on cultured cells. Generally speaking, this results in very good antibody staining. However, the cells tend to look markedly different than they did under phase contrast microscopy prior to fixation. This has my PI worried about how these images will be received by reviewers.

How does a methanol fixative work, and why does it cause a change in cell morphology? Are there parameters that can be adjusted to mitigate this effect?

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  • $\begingroup$ Give me one last bit of information, and I will throw up a re-write of why MtOH causes changes in cell morphology, why I really think you should be using acetone, and the unmasking information I already put up. What are you culturing the cells on, and what are you fixing them on? Is it a PP chamber slide, trans-well, what? Direct trouble shooting a specific problem is a cornerstone of SE sites, check out stack overflow if you haven't looked at it already. If you want a more general answer, be prepared to show what research you have already done to answer the question yourself. $\endgroup$ – Atl LED Nov 21 '14 at 16:54
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Yes, fixation of almost any kind can have effects on morphology. When you take a free flowing, protein spiked fat-blob (ie cell membrane), and make it rigid, you are going to get some differences.

A fun visualization of this can be done by wrapping cellophane/shrink wrap around a serological pipette and dipping it in a dry ice and methanol bath. It's something I show students all the time before getting into histology work.

Back to what you need. What cells are you using and what are you growing them on? Have you tried acetone fixation and you know it destroyed your plastic? The amount of time and concentration needed for acetone fixation is low enough that most modern chamber slides can handle it. Do you need the membrane to be permeabilized in fixation?

I don't know the main effect you are measuring, but assuming you have good controls, you should be able to see it past the fixation. The effect in your controls should be similar to your treated groups, unless the treatment is specifically effected by your fixative.

Moving to some more general recommendations. I think you should reconsider the fixative’s you’ve ruled out, because I am unconfident about their application. The base source I always recommend students to turn to is the Wiley "Current Protocols in X" book that is relevant to the task at hand. In this case, I think the most relevant text is Current Protocols in Immunology, and the appropriate unit is 21.4 "Immunohistochemistry."

I think a particularly useful, and relevant to this question if you are concerned with formalin masking, is Table 21.4.3.

enter image description here

That is for human antigens, but the “antigen retrieval” process works for murine cells in most cases. You are right that you antigen could be blocked by formalin, but the solution is often unmasking as MtOH are often more harsh to morphology. To support protocol 1:

Formalin or paraformaldehyde fixation cross-links protein, often making antigens inaccessible to specific antibodies. The antigen retrieval protocol, which combines high temperature with buffers at different pH, has been very effective in unmasking antigenic determinants which may be masked in fixed tissue or fixed cell preparations, particularly determinants within the nucleus or cytoplasm (Taylor and Shi, 2013).

My modification of the protocol is:

Sodium citrate buffer: 0.1 M citric acid/0.1 M sodium citrate, pH 6.0

  1. Wash sample on a liner rocker for 5 min w/2x culture volume of d.i. H2O
  2. Place your slides in 1mM citrate buffer in Coplin jars with the lid slightly off
  3. Weigh the jar in final state so you know how much water to add back
  4. Microwave 4-7 min. You don’t want it to go to a boil, and you need to remember to allow for venting
  5. Weigh the jar again and add back water
  6. Microwave another 4-7 min, again you don’t want a boil
  7. Take out of the oven and allow it to come to room temp by sitting it on the bench. If you do it too fast you’re going to disrupt the membrane.
  8. After it’s back to room temp, wash 2x PBS+0.05% Tween20 for 10 min each
  9. Try your IHC again, it will probably work now.
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  • $\begingroup$ It's a surface antigen on endothelium, so I'm trying to leave the membrane as intact and possible. Acetone ruins our culture vessels pretty quickly. How long do you fix for? I am generally against the use of kits. They're too much of a black box (one of the many reasons I dislike the growing role and dubious business practices of some vendors). If you check the MSDS on that life technologies kit, you'll see that the fixation medium is just formaldehyde. Which I have already tried once, but am going to give another go tomorrow. $\endgroup$ – Chastain Nov 21 '14 at 3:27
  • $\begingroup$ @Chastain First, you should almost never be using straight formaldehyde on an endothelium. You should be using a buffered formalin, about 10% in concentration. Second what's the tissue source? (Animal/organ) $\endgroup$ – Atl LED Nov 21 '14 at 12:49
  • $\begingroup$ I will also note that side by side with a buffered formalin and tissues from BALB/c mice, we saw much better preservation with the kit. Could just be how they are buffering things. I will post up the MtOH and Acetone protocols I use after my morning meetings. $\endgroup$ – Atl LED Nov 21 '14 at 12:57
  • $\begingroup$ Thank you. It's murine and isolated from several different organ systems. To be clear, the question is not about tissue section morphology. Morphology of sectioned and stained tissue is fine. This question is specific to cultured cells. Which is one of the reasons that it is so frustrating. $\endgroup$ – Chastain Nov 21 '14 at 13:40
  • $\begingroup$ This perhaps could be settled out better over chat. Histology, fixation, and IHC are things that have to be highly optimized. My staining of primary human lung epithelium was tweaked over several years and labs. I understand you are trying to fix cultured cells. Is it a monolayer or transwell system? From the sound of your last comment, you are co-culturing multiple types of cells (as they are from different tissues). Are they primary or imortalized? I'm trying to get you a solution rather than a general answer on fixation. If you don't want 1, I can write up an in-depth answer on fixation. $\endgroup$ – Atl LED Nov 21 '14 at 15:42

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