Try to use10% buffered formaldeide for 18 hours fixation the maximum

Dear Anne Marie Gannon, so you like us to guess a little bit about what you are really requesting. From your post we know that it is rodent (rat?) uteri in which you have to localize first the area / implantation sites and seeing them by means of stain application to cut out oriented for further processing (fixation for LHistology?, LM, EM?[morphological studies], Fluorescence methods???). It would be also interesting which method you use for staining, guessing that "ammonium sulfide" will be a / one component of a certain staining solution...?? Please specify your problem a little bit more precise, help or any recommendations then also will be mor precise...Best regards,

Wolfgang, I want to count implantation sites and then fix for histology (H&E), but ammonium sulfide renders the tissues unusable for histological fixation. I am asking what solution I should use instead of ammonium sulfide to preserve the usefulness of the tissue for histology.

We used an iv injection of 1% Evan's Blue to localize implantation sites. (See my publication "Lack of Effect of Hind Limb Suspension on Early Embryogenesis and Uterine Implantation in the Rat"). The sites were then processed for paraffin embedding and sectioning with H&E staining. Note that the only difference from the paper I would suggest is to use Bouin's fixative as opposed to the formalin. We had better morphology with the Bouin's.

Dear Anne, apologize if I again am guessing....

but IF you use the following method for staining, please let me know...

citing ammonium sulfide (only) as reagent for staining implantation sites was found @ http://www.ncbi.nlm.nih.gov/pubmed/9408976 :

Teratology. 1997 Oct;56(4):252-61.

Critical period of carbon tetrachloride-induced pregnancy loss in Fischer-344 rats, with insights into the detection of resorption sites by ammonium sulfide staining.

Narotsky MG, Brownie CF, Kavlock RJ.

Source: U.S. Environmental Protection Agency, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Research Triangle Park, North Carolina 27711, USA. [email protected]

=citation=:"Non-gravid uteri were examined for resorption sites, placed in 10% ammonium sulfide, and re-examined for stained resorption sites approximately 1 and 4.5 hr later. ..... Ammonium sulfide staining clearly yielded a more accurate account of the incidence of FLR. The technique was most effective when the staining period was extended to 4.5 hr, as two cases of FLR were revealed that had been undetected after 1 hr of staining. " [nofollowing histo- technique or images of histology sections or staining] =End of citation=

Another article would be:

http://www.ncbi.nlm.nih.gov/pubmed/2580727

Jikken Dobutsu. Experimental Animals [1985, jan,34(1):17-22]

[Studies on implantation traces in rats. I. Size, observation period and staining].

[Article in Japanese]

Yamada T, Hara M, Ohba Y, Inoue T, Ohno H.

=Citation=: "All kinds of implantation traces were stained distinctly with 10% ammonium sulfide, 0.2% sodium hydroxide and 2% potassium ferrocyanide. In this staining method, sodium hydroxide has an excellent effect on the staining of implantation traces. Specimens washed in water after being stained with sodium hydroxide and fixed in formalin can be preserved without discoloration for a long period of time." =End of citation=.

also found:

Yamada T, Hara M, Ohba Y, Inoue T, Ohno H: [Studies on implantation traces in rats. II. Staining of cleared uteri, formation and distribution of implantation traces]. Jikken Dobutsu; 1985 Jul;34(3):249-60

=citation:= This staining method was convenient for observation of the implantation traces.

Though the implantation traces in rats which had been delivered four times were arranged like beads, they were recognizable by NaOH stain as either old or new traces, the former appearing smaller without agglomerate cells and a little more brown in comparison with the latter. =end of citation=

another source:

"staining the uterus with ammonium sulfide or sodium hydroxide (Salewski, 1964 * ; Yamada et al. 1988 **)

- *SALEWSKI VE (1964): Faerbemethode zum makroskopischen Nachweis von Implantationstellen am Uterus der Ratte, Nauyn-Schmiedebergs Arch. Exp.Pathol. und Pharmak. 247, p 367ff.

- ** Yamada T, Ohsawa K, & Ohno H (1988): The usefulness of alkaline solutions for cle...

A traditional method for identification of early implantation sites if the Pontamine Sky Blue reaction (also known as Chicago blue) - inject 0.1 ml of the 1% blue dye solution through a tail vein and leave for 3 mins - the implantation sites are easily identified as distinct blue bands along the uterine horn. Excise tissue and fix as per normal.

Thank you very much, Vera!

Some previous suggestions (and questions) seems to be very useful. What is the aim of your study - do you want to localize rat ? nidation (implantation) site only? In case of histology method like immunohistochemistry - it is useful to use 10% buffered formaldeide for 18 - 24 hours according my experience. We had better morphology with the Bouin's (especially in fine sensory organs fixation) - I agree with Joan, but in this case is immunohistochemistry using questionable. Best regards.

I second what Vera has suggested. I have used it often for visualisation of rodent implantation sites. I struggled a bit with the injection into the tail vein so once the animal has been anesthetized I make a ventral midline incision. I then inject 1ml of 1% Pontamine Sky Blue into the inferior vena cava. You can immediately see if the injection was done correctly as the entire vessel system turns blue.

We generally fallow this method.

Subject the uteri that appear non-gravid to Salewski staining for the detection of implantation sites that are not visible macroscopically to confirm the non-pregnant status a.

 Staining solution for non-gravid uterus: For identifying implantation sites

a.   Ammonium sulphide: 10% (vol/vol) solution, commercially available.

 b.   Hydrochloric acid: 1% (vol/vol) solution (1.0 ml of HCl in 99 ml of deionised water).

 c.   Potassium  ferricyanide:    2% (wt/vol) solution (2.0 g of potassium ferricyanide in 100 ml of deionised water).

Staining procedure

Place the opened uterus in 10% solution of ammonium sulfide for 10 minutes and rinse with tap water.  Then place it in a solution containing equal parts of 1% hydrochloric acid and 2% potassium ferricyanide for 10 minutes. Implantation sites if present stain bluish black indicating pregnancy.

Hi Anne

I agree with Mr. Vera Krizkova  in using Evans blue dye, it is very easy and sufficient 

look at this paper.

http://www.japsonline.com/admin/php/uploads/1379_pdf.pdf

Good luck

pottasium ferricyanide also can be used