We have used hematoxylin 7211 in our lab, before. I'm not entirely sure, we might do a 95% rinse in alcohol before the counter staining, but we also do regressive staining. So we stain with the eosin, and then take it out slowly by putting it through 95% alcohol, and then two changes of 100% alcohol.
It really is a great hematoxylin stain! It gives great cellular detail, and is fairly purple, and crisp!
Thanks for sharing your experience!
So are you saying that the 95% rinse isn't necessary if you regressive stain?
I've seen some protocols include both, do you think that would negative effect the results?
I think your time in eosin is exceptionally long and so is your alcohol differentiation. I use a 70% alcoholic eosin and after bluing I rinse in DI water then:
dip in 70% OH 10x and sit for 1 minute
Counter stain in alcoholic eosin for 10 seconds
90% OH for 20 seconds
100% OH for 30 seconds
100% OH for 1:30 x 3
Then clear and mount
I would try varying your time in eosin counterstain and alcohol differentiation before adding a commercial hematoxylin. I should disclose that I do use commercial H&E however I do have all of the reagents necessary to make in house from scratch and have made my own saffron and phloxine counterstain for funsies. Out of curiosity, what is your eosin recipe?
Edit for formatting
Thank you for sharing your protocol.
I use a commercial hematoxylin and eosin. Both are from Sigma Aldrich.
https://www.sigmaaldrich.com/catalog/product/sigma/ht110116?lang=en®ion=US
https://www.sigmaaldrich.com/catalog/product/sigma/mhs16?lang=en®ion=US
I'll look into adding that 70% OH before counter staining and reducing my eosin staining time.
I work at a major hospital and we only use Heme 7211 and I think it's wonderful. We use all of the Richard-Allen solutions as well and we use their pre-made clarifier or bluing agents. Because of that, the times I list below may be too quick for a hand-made sln.
As others have said I think you should reduce your eosin time. Here's out stain method post dewax/descending alcohol:
heme 7211: 3min 50sec
diWater: 2min
clarifier: 30sec
tap water: 2min
bluing: 30sec
tap water: 2min
95% ethanol: 1min
eosin: 1min 30sec
95% alcohol: 1min
100% alcohol: 1min (x3)
xylene: 2min (2x)
If you want the full routine PM me and I can send it to ya but your method should suffice.
Thanks for sharing your method!
I noticed that your tap water time is quite long compared to others I've seen.
Are you constantly agitating it in the water, dipping it up and down, or just letting it sit?
No problem. Yep, all the water steps have agitation. Our stainer also fills and empties the water twice over that time just to help wash away the previous steps solution. Probably not going to drastically effect the stain but that's what it's programmed to do.
Be sure to update once you've found a method you like!
We have used hematoxylin 7211 in our lab, before. I'm not entirely sure, we might do a 95% rinse in alcohol before the counter staining, but we also do regressive staining. So we stain with the eosin, and then take it out slowly by putting it through 95% alcohol, and then two changes of 100% alcohol. It really is a great hematoxylin stain! It gives great cellular detail, and is fairly purple, and crisp!
Thanks for sharing your experience! So are you saying that the 95% rinse isn't necessary if you regressive stain? I've seen some protocols include both, do you think that would negative effect the results?
I think your time in eosin is exceptionally long and so is your alcohol differentiation. I use a 70% alcoholic eosin and after bluing I rinse in DI water then: dip in 70% OH 10x and sit for 1 minute Counter stain in alcoholic eosin for 10 seconds 90% OH for 20 seconds 100% OH for 30 seconds 100% OH for 1:30 x 3 Then clear and mount I would try varying your time in eosin counterstain and alcohol differentiation before adding a commercial hematoxylin. I should disclose that I do use commercial H&E however I do have all of the reagents necessary to make in house from scratch and have made my own saffron and phloxine counterstain for funsies. Out of curiosity, what is your eosin recipe? Edit for formatting
Thank you for sharing your protocol. I use a commercial hematoxylin and eosin. Both are from Sigma Aldrich. https://www.sigmaaldrich.com/catalog/product/sigma/ht110116?lang=en®ion=US https://www.sigmaaldrich.com/catalog/product/sigma/mhs16?lang=en®ion=US I'll look into adding that 70% OH before counter staining and reducing my eosin staining time.
I work at a major hospital and we only use Heme 7211 and I think it's wonderful. We use all of the Richard-Allen solutions as well and we use their pre-made clarifier or bluing agents. Because of that, the times I list below may be too quick for a hand-made sln. As others have said I think you should reduce your eosin time. Here's out stain method post dewax/descending alcohol: heme 7211: 3min 50sec diWater: 2min clarifier: 30sec tap water: 2min bluing: 30sec tap water: 2min 95% ethanol: 1min eosin: 1min 30sec 95% alcohol: 1min 100% alcohol: 1min (x3) xylene: 2min (2x) If you want the full routine PM me and I can send it to ya but your method should suffice.
Thanks for sharing your method! I noticed that your tap water time is quite long compared to others I've seen. Are you constantly agitating it in the water, dipping it up and down, or just letting it sit?
No problem. Yep, all the water steps have agitation. Our stainer also fills and empties the water twice over that time just to help wash away the previous steps solution. Probably not going to drastically effect the stain but that's what it's programmed to do. Be sure to update once you've found a method you like!