I am doing IHC with bone and cartilage samples (eg. growth plate).
For antigen retrieval, I usually heat the samples in Citrate buffer (pH 6.0) using pressure cooker (for 1 min at max. temp and pressure and cool down for 20 min.)
But, bone and cartilage part are peeled off from the slide in high temperature.
In case of proteinase K - induced antigen retrieval, tissue is okay, but it doesn't work, no staining.
Any better antigen retrieval method for bone or cartilage sample?
Hi,
You could try the citrate buffer in a microwave/waterbath, and keep it just below boiling temperature. You will have to find out what the perfect settings are by trial and error. 10 minutes should do the trick.
We use a PT-link by DAKO for antigen retrieval. That heats the buffer to 95°C.
With kind regards,
Ronald
Thank you for your kind answer, Ronald.
I am using pressure cooker not to buffer get boiled under high pressure.
In my experience, bone and bone marrow part easily detached from slide only at 80-90°C.
( They are decalcified samples)
So I am trying not to heat the samples, but doesn't work as HIER.
I Baked samples at 55°C, washed only with PBS but I cannot manage sample detachment.
I would try coating your slides in gelatine first to make sure that the samples really stick to your slides or would invest in coated slides for IHC. I use a pressure cooker with Citrate buffer as well and rarely have the tissue slide off.
Good Luck
Thank you Mr. Turkewitz.
I would try other slides.
and I would appreciate if you recommend good adhesive slide or company
which widely used for bone study.
Best regards.
Hi Bong,
I would recommend Agilent's coated slides for IHC. They work very well with all types of tissue as well as Leica's adhesive charged slides.
Best of Luck
https://www.agilent.com/en-au/products/immunohistochemistry/ancillaries-for-ihc/dako-pen-slides-pascal-quality-strips/ihc-microscope-slides-flex
https://www.leicabiosystems.com/histology-consumables/slides/adhesivepositively-charged-slides/
Hey there,
we also work with cartilage and decalcified bone samples. These samples even detach by room temperature during non-IHC staining (safranin-O etc.). We found improved attachment on silanized slides, a coating which can be easily made in the lab and is a cheap solution. However, some samples were still washed off during IHC. Now, we use "Superfrost Plus" slides, which are quite expensive but result in successful outcome.
Best regards.
Hi everyone,
On the same topic, has anyone tried using a PT module (and citrate buffer) for antigen retrieval on bone sections please?
Thanks!
Kristina
- Badges
- Science method