I agree with everything that Valentina says. Duration of fixation is dependent on the size of the tissue sample. For very small pieces of breast cancer tissue (e.g. a core biopsy), 4 hours should be sufficient. For larger samples, overnight fixation may be required but I would not recommend fixing for longer than 24h and there MUST an excess of fixative. You must also pay attention to warm ischaemia time - the time between cutting off the blood supply to the tumour (or out of the patient/animal) and getting it into fixative. For most antigens warm ischaemia time must be kept to a minimum and it is crucial for phosphoproteins. There are plenty of publications in Pathology journals giving details of best practice for handling breast cancer specimens for routine examination of diagnostic and prognostic factors by IHC. Look out for authors such as Ian Ellis, Mitch Dowsett etc

I agree but it also depends with what are you going to perform, for example, for most of the IHC ideally you have to fix between 6 to 48 hours. If the size of the tumor is thicker and will need more time, it's better to cut the tumor while is still fresh, to allow a better fixation in all the tumor.

We do so, at least at first tissue (tumor) must lie in formalin 10%. Then you want to cut a piece of 0.3 mm and an area of ​​0.5 cm -1 cut a piece should lie in 10% formalin at least a day, and the formalin should be 10 times greater than the volume of the tumor. Then we do the wiring in isopropyl alcohol soaked in paraffin at a temperature not greater than 60 0C and make blocks for further cutting.

I would like to support Maria Soares's recommendation as there is evidence for it in relation to routinely tested breast cancer biomarkers including ER. PR. and HER2. As for the size of the tissue being it is important to attempt to slice larger tissue specimens down to to 1x1x0.5cm portions. It is important to regularly ascertain that any stored buffered formalin is at neutral or slightly above pH 7, before using it

I agree with Jacinto. Try not to prolong fixation in formaline more than 24 h but normally we make the inclusion of many samples to profit the inclusor capacities. After 24 h I change samples to 70% ethanol (I even didn't wash them previously with water) changing once to a fresh ethanol 70% solution after one week. This way you can keep them for years. Formalin fixation is the best to preserve tissues integrity but with time produces side effects of masking epitopes for IHQ. Ethanol is fixing by dehidration of tissues so there is a shrinkage of tissues and alteration of its normal appearance with very many artifacts. The combination of both is perfect under my experience.Put small pieces of tissue as Bharat recommends. Good luck!

6 to 48 hours!

Hi! I have a long experience with breast, prostate,lung and neuroblastoma tumours. I often use to perform IHC experiments and I can say that the perfect time for fixation is an overnight fixation or almost one day. I use 4% paraformaldehyde for fixation and as Valentina says you have to wash with PBS the tissues previously fixation. May please note that a prolonged fixation could ruin the epitopes and you could have a bad ihc reaction. Another suggestion is to put in agitation your samples with paraformaldehyde at 4°C.

Good luck!D.

It depends on the size of the specimen and on what you would like to look for.Generally 4-24 hrs is sufficient and I agree to washing in PBS after that or immersing in 70% ethanol,Most of the time these suggested procedures work very well.

Fixation time in formalin (4% buffered neutral formalin) requires small pieces and 24-48 hrs. It also depends from the tissue (ex. spleen should be fixed for at least 48 hrs). After fixation the pieces can be washed in tap water. In my experience fixation can be prolonged to several days without problems for immunohistochemistry, but antibodies must be paraffin tested. I would instead recommend not to store the specimens in alcohol.

Hi Shruok

I think all the comments are very useful and quite simsilar, so I am sure, now. you are going to make up your mind

We use to fix 24 hours to breast tumors.

Hi Shrouk

May I please suggest closing answers to this question as I pointed previously that most of the answers are similar and you can try any of them or all of them and then see the result to choose the best one with my best wishes.

Dear Simonetta, Is there are any problem in storing the specimens in alcohol? I'm already keeping my specimens in 70% alcohol.

May I refer you to the below reference including its abstract:

Validation of a Postfixation Tissue Storage and Transport Medium to Preserve Histopathology and Molecular Pathology Analyses (Total and Phosphoactivated Proteins, and FISH)

Michael M. Stumm, MD1,

Maja R. Walker1,

Caroline Stork1,

Noelle Hanoteau1,

Urs Wagner, MD2 and

Terence M. O’Reilly, PhD1

+ Author Affiliations

1From Oncology Research, Novartis Institutes of Biomedical Research, Novartis Pharma AG, Basel, Switzerland; and

2Head Pathology, Unilabs, Bern, Switzerland.

Address reprint requests to Dr Stumm: Guggeregge 6, 4303 Kaiseraugst, Switzerland.

Abstract

Tumor biomarker studies are integral to oncology clinical trials but may yield artifactual results owing to variation in sample procurement and processing. Ethanol, 70% vol/vol, was validated as a sample transport medium using markers of the PI3K/Akt/mTOR pathway. BT474 tumor xenografts were excised and slices were immediately placed into formaldehyde and fixed for 24 hours. Fixed tissue slices were immediately processed into paraffin or transferred to 70% vol/vol ethanol and stored at room temperature for 1, 2, and 4 weeks before further processing. Freshly cut tissue sections were evaluated for pAKTS473, HER2, pHER-2Y1248, pS6S235/236, and pS6S240/244, Ki-67, and HER2 by fluorescence in situ hybridization and stained with H&E and Masson trichrome. No significant changes were observed when comparing samples stored in 70% ethanol for up to 4 weeks with immediately processed tissue. Ethanol, 70% vol/vol, provides a safe storage medium for formaldehyde-fixed tumor tissue, facilitating sample transport during

I agree with Valentina, great tip!

Hi Shrouk, I work with mammary tumors of rats and we use 10% formaldehyde 6-18 hours. It is very important not to leave it more than 18 hours and to wash later in PBS for at least 30 minutes to eliminate the formaldehyde, since the latter could interfere in the IHC. Good luck!

Corina Sasso addition is in the same lines of most of the previous answers, so good luck and please start doing a pilot testing to see what sort of results you will obtain, good luck with my best wishes.

Dear all,

In answering Ahmed Mohamedani’s multiple „invitations“ to “close the question/thread” on this [e.g. “may I suggest closing answers to this question, etc. ] I nevertheless would like to add one post to this thread. Since I am interested in all kind of specimen preparation and fixation issues (as an electron microscopist I deal almost every day with such decisions like: which fixative, how long minimal - maximal, @RT or in the cold etc.).

Unfortunately only very late I got aware of the post by Shrouk El Khamisy (unfortunately this was not posted in Topics ,, or ).

I found some interesting posts/opinions on the matter (with some information I did not know about the practice out there 'in the wild') but wondered about how the terms for the fixative(s) used (i.e. 10% formaldehyde, 4% paraformaldehyde, 10% formalin) have been presented and commented/not commented on.

Ok, what I mean is: The original question by Shrouk El khamisy was: “what about using 10% formaldehyde?” (not knowing whether he meant 10% formalin or really 10% FORMALDEHYDE).

In some of the posts followed suggestions/recommendations for the following:

“Valentina Sala used 4% paraformaldehyde; Jacinto Costa: 10% formalin; Jacinto Costa: always used 10% formalin, but I will use 4%; Nikita Navolokin said: formalin 10%; Daniela De Martino uses 4% paraformaldehyde for fixation; Simonetta Astigiano: formalin (4% buffered neutral formalin) (small pieces) and last but not least, Corina Sasso: we use 10% formaldehyde…”

I just would like to point out that the percentages given and for the fixative Formalin and Formaldehyde were accepted mixed together without a tough clarification:

IF you use 4% paraformaldehyde (PFA) solution (either buffered or unbuffered) it should be / is clear for everyone WHAT you use. If you say: 10 % Formalin, you mean perhaps a dilution 1:10 of “commercially available (which is a trade name of MERCK Inc. and stands for a concentrated = min.38% v/v solution of formaldehyde… the latter originally is a gas, soluble only for 38-40% in water).

So in fact, 10% (buffered or unbuffered) FORMALIN is- with some deduction in terms of quality (see below) – the same as 4% (para-)FORMALDEHYDE solution (this said also especially for the post of Jacinto Costa: “always used 10% formalin, but I will use 4%”).

Use of “para-“formaldehyde (which sometimes is recommended for delicate specimens or very special IHC-immunolocalization procedures) implies the making of a “fresh” fixative solution from which is usually free from e.g. 10% methanol as stabilizer and CaCO3 powder for buffering the permanent formation of formic acid in the storage bottle due to chemical reaction mechanism in the solution over time of storage.

If someone uses this means – chemically correct – 2.5 times higher a concentration (compared to 4% hydrous formaldehyde solution, or, 10% Formalin, respectively - which usually is used in histology/IHC for tissue fixation).

So just to formulate the message:

10% (hydrous) FORMALIN solution usually equals 4% hydrous FORMALDEHYDE solution,

10 % (hydrous) FORMALDEHYDE (FA) equals approx. 25% hydrous FORMALIN solution.

Whether such a high concentration of FA can be used or would be of an advantage for / in tissue fixation suited for IHC-purposes I don’t know but admit that I have not found any references for such a task [only for fixing dead bodies in Pathology I know of such practice]

(cf. also postings: https://www.researchgate.net/post/Alternatives_to_formalin_fixation and

https://www.researchgate.net/post/4_paraformaldehyde_for_fixing_cells-4_degrees_or_room_temp )