Rabbit Brain with Kluver-Barrera Stain

discomorphella · Post by **discomorphella** » Wed Dec 17, 2014 3:37 am

It was just a matter of time really, until, like a hungry zombie, I got around to processing all the brain samples I've collected from my "hunter's histology" collection. This is the brain of the same jackrabbit that had its testes posted earlier (http://www.photomacrography.net/forum/v ... highlight=) so its getting to be a very well documented jackrabbit. Tissues obtained when the rabbit was cleaned, fixed in buffered formalin, dehydrated in 2-propanol, embedded in polyester wax, and sectioned at 7 microns. Which should have been 10 um or greater, I'll have to recut the block. The sections were stained using Luxol Fast Blue MBS (stains myelin blue) and Cresyl Violet Acetate (stains RNA,DNA etc., most cell processes some shade of violet). J.A. Kiernan's text on histochemistry has a good description on how K-B staining works. Basically the Luxol stains the myelin sheaths of neurons a deep blue, and the Cresyl Violet stains the neuron cell bodies. The photos were all taken using standard Koehler/brightfield (no DIC) on a BX-60, with either UPlanFlN 40/1.3 or UPlanApo 100/1.35 objectives, and recorded using a Diagnostic Instruments 2.5X adapter and a D810 operating in Live View (Mup) and EFSC mode. Or is it EFCS mode...photos were processed first using ViewNX2 and then further color balanced, downsized and filtered using ImageJ. A cross section through a mammalian brain has roughly 6 layers. You are probably familiar with the Grey (outer, the "switching layer") and White (inner, the "routing" layer to you EE's out there) matter. The white matter is a very dense collection of fibers, whereas the grey has several distinct layers of neurons.
I hope you enjoy the rabbit brain, next up is a fish brain, and then an elk. While this batch of stain is still good...

David

The first shot is one of the pyramidal cellular layers, which features a layer of more or less pyramid-shaped neurons (purple. with obvious nucleoli)

Here is one of the granular layers, which features round-appearing (in this staining scheme) neurons with a granular appearance on low-power shots, you can see there are a few blue-stained myelinated fibers above and below the neuron cell bodies

Next is a shot of the polymorphic layer, which has myelinated fibers of varying diameters and polymorphic (all manner of sizes and shapes) neurons

The 4th shot is a closeup of some of the polymorphic neurons and a tangled bunch of fibers next to them

The 5th shot shows the polymorphic layer transitioning to the white matter, now there are many myelinated fibers visible

The final shot is a cut through the white matter, which you can see is a very dense network of myelinated fibers with some cell bodies still visible

RogelioMoreno · Post by **RogelioMoreno** » Wed Dec 17, 2014 4:20 am

Interesting set!

Rogelio

escocat · Post by **escocat** » Wed Dec 17, 2014 5:17 am

A great job

discomorphella · Post by **discomorphella** » Wed Dec 17, 2014 10:20 pm

Thanks for the comments. I've added a few more images which show a cut through one of the ventricles as well as some lower mag views of the polymorphic region.
David

vasselle · Post by **vasselle** » Thu Dec 18, 2014 9:22 am

Hello
Très bon travail
Cordialement seb.

discomorphella · Post by **discomorphella** » Thu Dec 18, 2014 2:15 pm

Thanks. Now to start on the fish brain and then the elk. I really like the Luxol Fast Blue / Cresyl Violet stain combination.

David

Charles Krebs · Post by **Charles Krebs** » Fri Dec 19, 2014 1:06 am

Nice!
Visually fascinating (even though I know nothing about such brain structure

)

carlos.uruguay · Post by **carlos.uruguay** » Fri Dec 19, 2014 8:14 am

Great definition!

Pau · Post by **Pau** » Fri Dec 19, 2014 10:49 am

Excellent work, David

Nervous system is allways a very difficult subjet.
I most like the second image of each series because they show the ordered structure of the tissue.

I have some (bought) prepared slides that look pretty similar, is yours a standard staining technique?

discomorphella · Post by **discomorphella** » Fri Dec 19, 2014 11:07 am

Hi Pau,

You are correct, CNS tissue is typically more difficult to cut. Adding to the difficulty is the fact that for staining nerve fibers with techniques like Kluver-Barrera or the Cajal or Bodian silver methods, is the fact that to get a nice selection of fibers in the field of view, you must make a thicker section than usual. 10 or more microns instead of 5 or 6. This is because the fibers are going in all directions and you can't be sure of following one for very long with a thin section. But the thicker sections are more difficult to handle on the microtome.
This is a pretty standard staining technique. The Luxol Fast Blue / Cresyl Violet Acetate method was published by Kluver and Barrera in 1953 in the Journal of Neuropathology and Experimental Neurobiology. It has been modified over the years and I use the modification in J. A. Kiernan, "Histological and Histochemical Methods, Theory and Practice". You have to alter the differentiation step for both the LFB and the CVA for different animal's tissues. Even in these rabbit brain sections I think I "overdifferentiated" the Cresyl Violet, next time I will try to get the neuron bodies to stain darker violet. The fish brain (slides drying now) needed much less time to differentiate. So its a standard recipe but you have to do some experimenting for each batch of slides.
The interesting part about the Luxol Fast Blue stain is that not all of the myelinated fibers seem to take up the stain, so you can easily see some of the "routing" between layers, until you get to the white matter where the density is so high its just effectively a mat of stained fibers.

David

discomorphella · Post by **discomorphella** » Fri Dec 19, 2014 5:28 pm

Thanks Charlie and Carlos
This figure should explain the microanatomy that I didn't manage to get into one FOV at 40X objective FOV (well, at no X since I didn't get a cut (yet) that has all 6 layers in it).

The polymorphic layer is labeled the "mutiform layer" in this diagram. The far right diagram (taken from Ham's histology book I believe) is closest to the Kluver-Barrera stain in that the myelinated fibers are well defined.
I have posted shots of the internal pyramidal and granular layers as well as the polymorphic layer and a cut through one of the ventricles. The LFB stains red blood cells a shade of blue-green and the CVA stains the nuclei of the cells lining the ventricle violet like the neuron cell bodies. You can see a white blood cell (a violet-stained disk at about the very middle) in the vein cross section. The white matter would begin at the bottom where the fibers are concentrated.

David

GBS1 · Post by **GBS1** » Fri Dec 02, 2016 9:20 am

I've always liked the Kluver-Barrera stain. As a grad student I knew Betty Barrera who was a tech in our lab in her final year of professional work and I often greeted Dr. Kluver on his home at night. He was still working in his lab at the Univ of Chicago in his late 70's.

I'd like to use your images (in a Powerpoint) for a small lecture series and I want to know how to reference your work. I thought I'd would just include your name and whatever information you'd like me to include directly on the image.

I have other questions about the technical abbreviations that you use in describing your images but those can wait for some other time.

I think it might be helpful if you gave a bit more description. The top photos were of layers of the cerebral (or neocortex) and there was one of the CSF- generating choroid plexus of the ventricle.

Nice pics

discomorphella · Post by **discomorphella** » Sat Dec 03, 2016 7:57 pm

Thanks for your interest. The photos were almost all from the upper portion of the cerebral cortex as you surmised, if I had to guess without going back to the slide coordinates I would guess parietal cortex but I can't be sure. The brain had an ischemic time of an hour or so from when I got the rabbit to when it was cleaned and the brain was fixed in AFIP formalin, standard paraffin (paraplast plus) embedding technique and then cut at 6-7 um and stained using Kiernan's modification of the original KB method. Very standard histology. Please send me a private message and we can discuss what information to attach to the images, and also I can send you much higher resolution data; the forum is limited to 1024 on a side and these were taken at 36 Mpxl.
Best,
David

billben74 · Post by **billben74** » Sun Dec 04, 2016 6:43 am

Fantastically interesting post.
I interested in doing some zoo sections so really nice to see lots of details about this.

discomorphella · Post by **discomorphella** » Wed Dec 07, 2016 2:04 pm

I would highly recommend that if you have some chemistry or biochemistry or similar background, even just first year and organic chem, that you get a copy of Kiernan's histochemistry book, which has lots of well-tested recipes in addition to a lot of the theoretical background of histology.

https://www.amazon.com/Histological-His ... 1904842429

There are older books as well but Kiernan's text has some of the best and most up-to-date recipes and data. And then get some good safety goggles and high-quality nitrile gloves and start getting specimens. I typically get interesting tissues from hunting and fishing, and from what the cats catch in the barn (although once they caught a bat, which is bad news in the NW, as they can be rabid, THAT specimen I sent out to the state veterinary lab, the immunofluorescent reagents for rabies are expensive...).
Best of luck,
David