Section Thickness for Optical Fractionator and Immunohistochemistry

Stereology
#1

Hi everyone,

      I would like to learn how to decide the best appropriate thickness for both Nissl staining and immunohistochemistry (IHC). I will study learning and memory in the rats. We will run optical fractionator probes throughout the sections of animals. Sections for each animal will be used in both unbiased cell counting in the granule cell layer and in IHC for the ımmunopositivity detection of neurogenesis along the subgranular zone of granule cell layer in the hippocampus. So, as you might understand, the section thickness that I need to determine should be neither too thick for allowing the antibody to penetrate into tissue nor too thin for performing an unbiased cell counting. So, in my case, what should be the optimal thickness for both of them ?
#2

This is a tricky question because there is no right answer.

The thicker your tissue is, the faster you’ll be able to count it (because you’ll find – on average – more cells at each site with thicker tissue, you’ll need to visit fewer sites to sample the same number of cells). But, as you know, you can’t count a cell that isn’t labeled. This means that the ideal thickness for your tissue is the thickest it can be while still having complete penetration of the stain.

In practice, the best approach is likely to find someone else’s paper and copy the IHC methods they used, this will save you quite a bit of work in refining your staining methods.

#3

Thank you for your reply,

      As far as I know, the section thickness used for unbiased stereology may be somewhere between 40 and 60 micron. I don't take into account  the fact that the tissue usually shrinks after the procedures prior to mounting. Moreover, as time goes, the mounted section may shrink even more as a result of dehydration. I've been told by a colleague about an experience previously. In this case, the average mounted thickness was around 20 micron when the cutting thickness was 90 micron. I'd thought that amount of shrinkage was unusual. Do you think that the total shrinkage can be predicted given the cutting thickness ? What amount of shrinkage is acceptable for the rat brain slices ? 
     As to immunohistochemistry (IHC), when I start cutting the whole brain into slices, I think I won't be able to switch the cutting depth while performing. So, the whole brain of an animal will have been cut into the slices having exactly the same thickness. These slices will be used for both IHC and Nissl staining. Once the neurons stained with neural markers such as NeuN, I'll perform an immunoreactivity assessment using ImageJ software but not an unbiased stereology. Rest of slices will be used for Nissl staining using Cresyl violet., and then the unbiased cell counting will be done. I think I cannot perform an unbiased cell counting with the neurons fully-stained (axons and cell body) with immunohistochemical markers. Am I correct ?
    If so, as you suggest, I should look for optimal cutting thickness in papers in which the slices were used for both an IHC and a Nissl staining.

Kind regards,

#4

As far as I know, the section thickness used for unbiased stereology may be somewhere between 40 and 60 micron.

This is generally correct: your cut thickness should be somewhere around 40-60 (thicker is better, as long as you have complete staining/antibody penetration), while your mounted thickness should be a minimum of somewhere around 15 microns.

Moreover, as time goes, the mounted section may shrink even more as a result of dehydration.

Yes, exactly. Any alcohol/xylene washes during your tissue prep, as well as any time spent with the tissue exposed to air, will cause more shrinkage.

One thing folks do is to make sure that during fixation they are using PBS buffered formalin/PF at a physiologic osmolarity. If things are too salty, tissue will shrink during fixation due to water leaching.

Also, if you go to fast through dehydration steps, the issue becomes not getting rid of enough water before clearing (xylene/citrosolve) and subsequent paraffin embedding.

It’s definitely a good idea to track down someone else’s protocol and follow what they did.

I’ve been told by a colleague about an experience previously. In this case, the average mounted thickness was around 20 micron when the cutting thickness was 90 micron. I’d thought that amount of shrinkage was unusual.

That is pretty typical, especially if they were following a generic IHC protocol and not one that was optimized for stereology/the optical fractionator.

Do you think that the total shrinkage can be predicted given the cutting thickness ?

Because shrinkage is so dependent on the labeling/mounting protocol you use, you can’t predict the mounted thickness based solely on the cut thickness. But, shrinkage is generally very consistent – that is, as long as you use the same methodology, you can often expect around the same amount of shrinkage from subject to subject and experiment to experiment.

Having said that, even with the same tissue prep, certain experimental conditions such as young vs. old subjects or disease vs. control states can cause differences in shrinkage even with the same protocol. This means that you should never assume your final mounted thickness, always measure it during counting.

What amount of shrinkage is acceptable for the rat brain slices ?

There’s no answer to this question. More shrinkage usually makes it harder to count (you’ll need to visit more sites and do more work).

I think I cannot perform an unbiased cell counting with the neurons fully-stained (axons and cell body) with immunohistochemical markers. Am I correct ?

You should be able to do this. For the optical fractionator, you need a unique point which you will label. In your case, this would be the cell body, or rather the top plane of the cell body that comes into focus in the counting frame.

You would use the labeled axons only to help identify that this is a neuron that you are interested in counting, and then focus up and down to find the top of the cell body. With the top plane of the cell body in focus, use the counting frame rules to decide if this is a cell you can count or not:
You will mark this cell if:

  • If the current plane of the cell body that you are looking at touches a green line and does not touch a red line
  • If the current plane of the cell body that you are looking at falls completely inside the counting frame

You will not mark this cell if:

  • If the current plane of the cell body that you are looking at touches a red line anywhere
  • If the current plane of the cell body that you are looking at falls completely outside the counting frame
#5

Thank you for your detailed explanations, I appreciate it,
Sorry for this delayed response.

     I understand all of your points. You've made it very clear.

So, in my case, the cut thickness may be 40-60 micron.
As for staining technique, I don’t want to perform staining on
slides, as I think free floating results in a better penetration
compared to staining on slides. Besides, because the cut
thickness will be set around 40-60 micron, these thick slices may
drop off the slides while performing the staining procedure.

Do you agree with me on this ? What staining technique do you suggest me ?

Kind regards,

#6

I can’t recommend a particular staining technique – that is outside the scope of MBF’s support. The best strategy is to find a paper that does something similar and copy the protocol they use.

You can always email the authors of papers directly if you have questions.

I am happy to help field any questions you have on stereology technique or software usage, though.

#7

Thank you for your time,

      In the literature, papers don't generally reveal if the staining 
      having been performed either with free-floating or on-slide. 
      The authors just don't talk about that. They only talk about something 
      such as cutting thickness, incubation period, preparation of reagents, and any other 
       useful information regarding the staining protocol. Nevertheless, as you've said, the 
       only way to learn is to personally send an email to the author, asking if she/he used 
       the free-floating staining before.     
       

       Kind regards
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