Masson's Trichrome and MSB staining protocols

[@austinmorrissey5333]

Thanks for this video. Your recommendation on rinsing with acetic acid instead of incubating them improved my results significantly. Why is acetic acid used at all in masons trichrome? I'm having trouble noticing a clear difference before and after rinsing with acetic acid (at 4x and 10x on a light microscope).

[@damienharkin]

Hi Austin, The treatment with acetic acid should be brief and just enough to release the thick layer of Methyl blue from the surface of the slide. Two to three drops applied to the slide while held at an angle so that it drains across the section. Then flick off the solution from the slide before brief dehydration through two changes of absolute alcohol. If performing stain manually, you may wish to use a blotting step after the acetic acid, to improve the efficiency of the dehydration step. Acetic acid is used from memory because this is the solvent in which the Methyl blue is prepared. Thanks for watching D

[@austinmorrissey5333]

@@damienharkin That makes sense - thanks professor.

[@felipedeandradevieiraalves2265]

Hello Damien, thanks for this video, very informative. May i ask if you know an alternative option for the martius yellow dye?

[@damienharkin]

Interesting challenge. Tartrazine, picric acid and Saffron are all yellow dyes, BUT you would have to ensure that they are retained within the RBC throughout all the remaining staining steps. They may well be lost due to differences in molecular weight and solubility compared to Martius yellow. Have a go and let me know how it turns out.

[@felipedeandradevieiraalves2265]

@@damienharkin Thanks for the reply. I'll try it soon!

[@jameschan4964]

Hi Damien thank you for posting this video ... very informative. May I ask what is your post fixation protocol please? Bouin's @56C for 45 min to 1 hr? Is the temperature necessary? I have seen improve intensity of RBC uptake of Martius yellow. Thank you.

[@damienharkin]

Our standard is saturated aqueous picric acid at 60 degrees C for 45-60 minutes.

[@aaesuzuki]

what does it mean by "differentiation"?

[@damienharkin]

The term “differentiation” is used when applying a reagent to reduce background to a level where you can clearly differentiate between your intended staining target and non-target background structures.

[@Sabrina-ch1rt]

Hello Damien, I'm having issues with our MSB staining specifically on bone marrow trephines. Our staining protocol is similar but we don't post fix, and to be honest I'd never heard of that! Do you think the post fixation is a possible cause of our poor MSBs, do you have any other tips for MSB staining on BMT specifically? Many thanks.

[@damienharkin]

Hi Sabrina, post-fixation can make a huge difference if initial fixation is incomplete. Are you able to send me a few pictures?

[@Sabrina-ch1rt]

@@damienharkin Hello, thank you for the reply. Yes I will do when I'm back at work on Wednesday. Thanks again!

[@nyameyeakuffo397]

Hi Damien, I tried the Masson's Trichrome Staining on diabetic mice slide but I am not seeing any blue coloration to tell me the fibrotic nature in the glomeruli, is there a reason this happened? Is it because I rinsed the slide with 1% acetic acid?

[@damienharkin]

Hi Nyameye, Are you able to see any staining of connective tissue elsewhere in the section? If overall staining for connective tissue is poor, then it may well be due to excessive rinsing in acetic acid so you could try reducing this. e.g. no more than 1 mL applied the slide, then shake off remaining excess stain or blot. Some other options: (1) Are you are employing a post-fixation step in saturated aqueous picric acid (60 degrees C for 1 hour) prior to staining? This can improve the retention of methyl blue within the connective tissue. (2) You should also try the Van Gieson staining method covered elsewhere on my channel which is much simpler and provides adequate demonstration of connective tissue. If you require more detailed advice, you are welcome to join my channel as a Client Member. Client Membership enables you to obtain a written report on your staining outcomes based upon submission of images and protocols via email. Good luck either way!

[@nyameyeakuffo397]

@@damienharkin I was able to see collagen around the blood vessel but not in the glomeruli. I did 2 mL acetic acid in 198 distilled water .

[@damienharkin]

Is thickening of the filtration apparatus/basement membrane evident from performing an H&E stain? If so, then perhaps try staining using the PAS method rather than MT.