One thing that might be tried would be trimming out from the skull a block made of the pituitary gland together with surrounding structures (sphenoid bone and diencephalon). That block would be fixed in 10% neutral buffered formalin during 24 hours. After that, I think the gland should be hardened enough to try to extract it from the sella turcica. It could be extracted by careful chipping of the bony material with tweezers under a stereoscopic microscope.

I usually does not work with chicken, only human tissue.

Definitely I would try fixing first. If that doesn't work, cross sections can be made with a scroll saw, and the pituitary can then be cut away from bone with a razor. Always wear eye protection.

I do agree with first fixation and there after dissection. I may add intravascular fixation using buffered glutarldehyde, then procede to dissection using a concave spatula.

Dea Ferdinand, dear Jose and Matthew,

just my thoughts:

the "problem" reminds me of the work done in the late 1970es on rat brain(skull) preparation of whole hypothalamus... task: serial sections for reconstruction (semithin sections)...my starting point naturally was perfusion fixation (which is recommended here for several reasons, not only because of good preservation of structures). It will - naturally - depend also on the IHC to be done...

Not knowing whether you'll use "live chickens" in an experiment using animal it is not easy to advise for any preparation (dissecting steps, at least brain fixation first, or decapitation, peparing free the brain base and fix by immersion, then preparing the pituitary gland) mode of the brain/hypothalamic-pituitary gland region. I have been looking for any source of dissecting prep.steps for chicken ("Dissecting laboratory Manuals", such may be either very old or be part of the university "animal preparing" courses), but always found either perfusion fixation first and then detaching the skull peu-à-peu with the aid of dissecting forceps. You won't get rid of that manual step in this case. If the bony parts are too thick for breaking away with the dissecting forceps you could try to incubate the whole specimen ( after a sufficient period of immersion fixation, which will be approxim. at least over night @ RT) into a decalcifying solution as used usually in histology or histochemistry to get rid of the dense osseous material for proper sectioning. This naturally only can be done if such a decalcifying step does not interfere with your IHC...

The other question would be then which fixation solution would be best for the task you would like to achieve with IHC...

very best regards and good luck,

Wolfgang

Dissect the animal, locate the diencephalon,remove the entire hypothalamus from near the optic fibers, fix it in 10 percent neutral buffered formalin for 24 hrs,wash in running tap water for 24 hrs, dehydrate clean and then prepare the paraffin block, sections should be cut and stained preferably in Mallory's triple stain.

I have not worked with chicken before but guess the gland will be very small to detach from adjoining structures especially the bone. So my suggestion is you for 48-72hrs after which you decapitate and dissect the gland along side the bones which you then transfer to a decalcifying fluid. if you go with this, you may need a control to ensure the decalcifying fluid has no effect on the gland.

Alternatively, I have also transferred a goat head into liquid nitrogen to freeze it and then decapitated before fixing. It worked and the brain tissue was well preserved.

Good luck

Dear schmitt

First, make a fixation in 10 % neutral buff. formalin for at least 24hrs.

Second, extract it.

Third, do what you want.

I too have never worked on chicken but have worked on, or known people who have worked on, pituitary physiology in mice, rat, rabbit and starling. In all of these cases, the parietal and frontal bones are removed using small bone shears (or scissors). The brain is then rolled back to reveal the sella turcica (inevitably tearing the hypthalamic stalk). The diaphragma sellae is then split with the tip of a pink syringe needle and the pituitary removed by running the needle around and under the gland. This can be done fresh if required. The key is to split effectively the diaphgagma sellae otherwise the pituitary will be crushed as it is removed. As I get older, a dissecting microscope helps! I take the gland fresh for RNA extraction but it can be fixed for IHC. The gland becomes engorged with blood if the animal is stressed and especially if decapitated. This makes intact removal almost impossible. The more ethical approaches as given in Schedule 1 of the Animals (Scientific Procedures) Act 1986 are suitable and prevent this problem. Hope this helps!

Ferdinand: As a veterinary student, I used a tool used to dental pliars (used to extract teeth) to peel/chip all the bone off of a formalin fixed brain, optic nerves and attached eyeballs of an adult dog. What jose proposes is quite realistic.

Dear Ferdinand,

Also don't know about the chick but we work with the mouse pituitary gland and do ICH all the time on either paraffin sections or cryosections. The approach suggested by Ian Scott works just fine. I would just add: after carefully separating the pituitary from the surrounding tissue, use a plastic pipette to gently wash the pituitary with buffer. Repeat and keep freeing the pituitary with care from any remaining tissue attached to it. The pit will eventually float away into the PBS where you can then suck it into the pipette and then transfer it into your fixative solution.

I usually does not work with chicken, only mollusks and fish tissues.

I don't have experience with removing the pituitary gland, but I think the suggestions put forward by others sound good. I would only add that my favorite fixative for preserving structure without loss of antigenicity (since you will do IHC) is paraformaldehyde-lysine-periodate (Nakane's fixative). Attached is the original reference: McLean IW, Nakane PW. 1974. J Histochem Cytochem. 22:1077.

I have experience in removing pituitary gland of mice and rat. Our lab, we directly immediately remove pituitary gland from animals without perfusion to avoid the shrinkage of tissue. Then immediately immersed in the fixative.

I agree with Ayman ElKenay that fixation, without extraction, in neutral buffer formalin for 24 hours will impart some hardening to the soft pituitary tissue and thus facilitating its separation.

Dear posters all,

let me add the following:

first of all: great discussion, good hints for a practical solution Secondly: As I have learned "some years ago", all preparative work on such sampling (very tiny parts of a chicken brain) be recommended to be done with the aid of a / under a dissecting scope (at least magnification x=times 5-10). All primary preparative steps to "harvest" the gland under permanent flushing of the < excavation site > with fresh fixative. I guess, elapsed time from start of prep. getting out the whole pituitary into the fixative will count for physiological and structural preservation reasons.

If hypothalamic region(s) will not be used for the IHC section study or it is not necessary to preserve these, one could cut away most of the upper hypothalamic tissue [which, I think, was proposed by B. Jha above, and: not knowing whether the diencephalon/hypothalamic region/brain as a whole can be rolled back in chicken, as Ian Scott has proposed) leaving only a small basal brain tissue area (at least the hypothalamic stalk ) intact]. The whole preparation procedure until a first application of fixative from a pipette should not take more than 1-2 minutes, I guess. I remember that with the rat hypothalamic regions I had to prepare (from puppies 1d to adult rats 360 days of age) in former days I started with approxim. 7-10 min for ending up with the total exposure of brain without any bony part of the skull ....after some time this was reduced to 1-2 minutes...... ok, the brain still previously was fixed by perfusion fixation already....and commenting Wiphawis post, again: a perfusion fixation would be recommended (if it would help anyway or be feasible) since shrinkage or swelling of (brain) tissue due to a perfusion fixation depends on parameters one can control for sure (perfusion pressure, temperature, tonicity [and pH] of fixative). I remember also the use of some kind of anatomical pliers (proposal Jose Mario) to spread apart the bony skull more rapidly (and to add fixative into the cleavage sites). Other parameters to be thought over would be the right fixative (as has been said also by others, coming into mind besides buffered formalin and McLeans PFLP, as proposed by Gerald, also kind of Bouin's modifications = picric acid added) . Also it should be checked whether anesthesia of animals would interfere with hormonal / physiological functions in the pituitary gland....

If anyone should find a sketch of chicken brain morphology or get knowlegde of a Dissceting Laboratory Manual I greatly should acknowlegde to post this here too...

All best wishes to all of you,

thanking for great discussions this year (most of the posters I know +/- well), have a nice Christmas time (if you will celebrate) and a Happy, healthy, successful and prosperous New Year,

Wolfgang

Hi to all of you! Thanks a lot for this very big and enthusiastic help! I really have to sit down and think of all the options you gave me. As we don´t have the possibility at the monet to do the perfusion technique we probably will go for the decal method (cutting as much bone as possible and then decalcify the rest and dissect the pituary). I will let you know how it worked.

Merrry Chrsitmas and a Happy New Year to you!

On my opinion the best fixatives for pituitary glands are" Heidenhain's "Susa" and bouin.After fixation during 24 hours the samples'd be moved to ethanol 70%."susa" has decalcification effect that is important in your case.

Heidenhain's "susa":

Dist.water 80ml

Formalin (38-40%) 20 ml

Sodium chloride o.5 g

Mercuruc chloride 4.5 g

Trichloracetic acid 2 g

Glacial acetic acid 4 ml

Happy new Year!

Get is as fresh as possible, make a nick -bivalve incision with a sharp new blade along the stalk and fix in neutral 10$ buffered formalin at room temperature for not less than 8 h (ideally 16-24h) and then process it standard way. This is a very standard method, works well for any IHC (depending of course on your primary antibody) on human and animal tissue samples.

in order to separate it from bone you need to destroy "sella turcica" i.e the niche where the pituirary is sitting. As the chicken brain and skull is notoriously small you will need to use magnifying glasses or a dissection microscope in order to get a good sample out.