Hello everyone!
I´m recently working in a biochemistry-lab as a medicine student and I´m doing immunostaining on mice brain´s.
However, the slices I´m treating will look kinda good in the first few washes, but towards the end (especially after HCl-treatment), the probes will shrink and fold together, so that it´s almost impossible to mount them properly.
Maybe anyone with more expertise can give me input about some common mistakes I should try to avoid.
Thank you guys very much!
Hi Miray,
I think very thin tissue sectioning and different reagents for antigen retrieval steps may help (such as in this publication Article Differential microstructural alterations in rat cerebral cor...
). Additionally, you can also reduce the time for dehydrating steps of tissue sections. We measured tissue shrinkage of thick tissue sections of the hippocampus of the Macqaque brain and, observed maximum shrinkage in the thickness of the tissue and a little shrinkage in X and Y dimensions too. You can see this article.Article 3D structure tensor analysis of light microscopy data for va...
).
Try to get: M.A.Hayat (2002) Microscopy, immunohistochemistry and antigen retrieval methods. KluwerAcademic/Plenum publ. NY. or Boon Me, Kok LP (1994) Microwaves for immunohistochemistr Micron 25:151-170; Kok Boon 1992 Microwave Cookbook for Microscopists Coulomb Press Leiden. Moreover in Methods an extra volume has been dedicated to immunohistochemistry.
Succes. Marani
thank you guys! I will definitely check out the articles and books!
Hi Miray,
I've few questions for you before giving an answer
(1) Are the tissues paraffin fixed and slicing them on microtome?
(2) How are you coating your slides before taking tissue on them?
However, possible reasons could be an inappropriate coating of the slides and/or embedding in wax is not perfectly done! Tissue processing matters a lot in histology...
Don't hesitate to inbox me. All the best!
It is referring to tissue not included in a pattern, such as paraffin.
Then it will depend at first, of the type of cutting method that you use, freezing microtome, vibratomo.
In the first case, the tissue had to cryoprese very well, so that there are no problems in court. It is also important, the temperature of the tissue, the blade and the placement of the tissue n the equipment, any of those variable, can cause the wrinkle cut and be lost.
In the second when Vibrato is used, the ideal is to make the cut once the tissue has been fixed, although a good cryipreservation process, will keep the quality of the fabric, if you want to group it, to make a single work session.
In this method, fabric fixation is very important so that the consistency is adequate, the position of tissue on the equipment, which the blade is new, these blades are very economical so they can be changing frequently.
In that case one should tie the tissue that floats in the buffer solution that is used, so if the cut was done correctly, the tie of the tissue is very simple and can be rectified.
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