methylene blue .05% staining protocol for aberrant crypt foci? for colorectal cancer
Here is the protocol I used in my work;
After the abdominal cavity was opened, the gastrointestinal tract
was removed from the stomach to the rectum and flushed with
saline. The entire length was opened longitudinally, fixed in
2% formalin, stained with methylene blue and examined under
a stereoscopic dissecting microscope.
How to make it:
1. Make 0.5% Methylene Blue Stain by dissolving 15 mg Methylene Blue (Sigma-Aldrich #M9140) in 30 mL of distilled water. Prepare two more containers with only distilled water in them for washing.
2. Stain the G.I. by immersing in 0.5% Methylene Blue for 30 seconds.
3. Rinse in first container of distilled water.
4. Rinse in second container of distilled water.
5. Place G.I. back on paraffin block with 70% ethanol.
6. Examine the stained colon with dissecting microscope ( or Stereoscope) for aberrant crypt foci or any polyps (tumors). ACFs are easily identified after the MB staining under a scope.
https://www.ncbi.nlm.nih.gov/pubmed/23035966
thank you very much its very helpful answer, can u please explain the fifth step in more details ? thanx again
https://www.researchgate.net/profile/Muhammad_Aslam21
Step 5 (more personalized approach): I use paraffin blocks to fix mouse / rat colon (after opening it longitudinally) with formalin while pinning it down to block overnight to keep mucosal surface up as colon is easily curls and make it difficult to look under a stereoscope. After 24 hours discard formalin and replace it with 70% Ethanol for another 24-48 hours till you are ready for staining the tissue with MB. Once I stain it with MB, I usually add PBS or saline or even water or ethanol to keep colon tissue moist. If it gets dry then that will effect histological analysis difficult. If you stained it with MB and not ready to examine it right away with using a stereoscope then please don't use ethanol after introducing MB as it will reduce staining effect. I identify tumors (micro adenomas) or ACFs under scope and cut it for histology as soon as possible.
Once the colon tissue is stained with MB and has been examined under a microscope, how do you remove the MB stain to prepare for histological examination?
To remove MB, you can gently rinse the colon tissue with 70% Ethanol (or PBS) and put it in clean 70% Ethanol till you submit the tissue for histology.
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