Hello everyone. I'm having a little conundrum in my lab. We've been running our h&e staining as usual and all of a sudden, slides present this "bluish" "smudged" area. It mostly occurs in glandular cells of colon, cartilage. We have tried troubleshooting it and:
- it is not the staining, as we had an external slide, and staining is perfect (colon)
- it is not a fixation problem as specimen are fixed long enough from 6hrs(small bxs) to 48hrs (resection post opening)
We are thinking it might be a processing issue, maybe the dehydration step, maybe a poor quality of alcohol (we use IPA).
Please see attached 2 bad staining from a colon bx and neck resection, and the external colon slide.
Hope someone could help.
Regards
AA
it seems to me that the tissue was folded and that is why the staining is faulty. I guess that tissue was not accurately placed into a cassette for paraffin embedding. Inaccurate placement led to folding and associated problems with staining.
I agree with Olga. Some tissues including cartilage do not interact with the glass well and for such tissues, a modified glass surface is recommended. Please read this for a full understanding: https://www.leicabiosystems.com/us/knowledge-pathway/glass-slides-science-of-surface-modification/
Thanks both for the reply. I'd make note of all your suggestions. Can you also look the the third image. You can clearly see that the glandular cells are blue, there is no nuclear stain. Looks as the stain is washed out. Can you help me with that please?
Image 3 is a sample dissected and processed in house, image 2 is an external slide. Visible the difference quality of stain.
What you described as "glandular cells are blue" ( image 20221201_1002
56) is about the nuclei of those cells. the nuclei are just very large and you confused them as cells. I do not see a problem with the staining. All is good. the large nuclei and pink cytoplasm around them. the " blue" structures are not " cells", it is the nuclei.
Olga is correct again. The stain density is lower in the epithelial cells as the genetic material is not as densely packed... your staining here is good.
There is a mild staining issue in the third image in the 2 o'clock and central 2 o'clock directed areas, they are small and probably neglectable but if widespread throught the whole slide I'd recheck the staining procedure (processing issue is unlikely).
That's exactly the issue!!!
It's been happen ONLY with colon and rectal biopsies. All other tissue aren't affected.
Protocol has been reviewed, an external slide was stained (image 2) with no issues at all.
That's the reason why I was questioning the processing.
If it happens in the biopsies only I would probably try a protocol for small tissue samples. There are many different open source protocols available I just cannot say which one is better.
You cannot expect the whole slide to be absolutely perfect. I suggest using the current protocol as any deviations may deliver worse outcome. You never know beforehand. Be aware: when you try a new protocol you are not necessarily improve the staining. However, you may need to be careful when placing tissue into an embedding frame/cassette before submerging the block into paraffin. careful, accurate placing of the tissue will deliver a better sample for future cuttings. people are using few tricks to ensure the perfect location of the tissue in the block, please read this protocol here Tissue Processing Overview: Steps & Techniques for Histopathology (leicabiosystems.com): Steps & Techniques for Histopathology (leicabiosystems.com) or here Histology Tips | Penn Center for Musculoskeletal Disorders | Perelman School of Medicine at the University of Pennsylvania (upenn.edu)
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