[Histonet] Thionin Staining Recipe and Protocol
Good Morning Histonetters! I am looking for advice on my thionin staining recipe and protocol. The recipe I use currently ends up being just under 1% Thionin. It is made by mixing 80ml 1M acetic acid and 14.4ml 1M NAOH to a pH of 4, and then adding 305.6ml 1.3% stock thionin. When I used this protocol last, I rehydrated the mounted rat brain sections (perfused with 4% buffered para) through ETOH to dH20 steps, then left the slides in thionin for 6 minutes, followed by 2x2min rinses in dH20, 1 min in 70% ETOH, 3 min in 95% ETOH, and finally absolute ETOH and xylene for coverslipping. The tissue came out too dark, and what I am wondering is if I should change the thionin recipe to something a bit more dilute, or if I should focus on the baths following thionin, for instance only rinsing in water and moving into a .1% acetic acid in 70% ETOH differentiator? Any help, either by directing my own troubleshooting or even recipes and protocols would be greatly appreciated! Thanks, as always, for being such wonderful resources for those like myself who have such little histology background :) Amanda Madden ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Thionin Staining Recipe and Protocol
Simple CV or Thionine method: Cresyl Violet or Thionine0.1 gram Distilled water100 ml when dissolved add 10% acetic acid0.5 ml *or * glacial acetic acid100 µl 1. Bring paraffin sections to water as above, frozen or cryostat sections are rinsed in water. 2. Stain 2-10 minutes in 0.1% cresyl violet or thionine. 3. Rinse in 2 changes of distilled water, 30 seconds to one minute each. 4. Dehydrate quickly in a few dips in 70% ethanol then 95% ethanol, 15 seconds, then 2 changes of 100% ethanol, one minute each. 5. Clear in 2 changes of xylene and coverslip with DPX mounting medium. Some stain will come out in the lower alcohols which is normal. Only nuclei and Nissl should stain. Sounds like your stain is too concentrated. OR, if you prefer a buffered solution ... 1. Bring paraffin sections to water. 2. Stain 20 minutes in: 0.1% aqueous *Cresyl Violet* or *Thionin*, one part. 0.2 M Walpole acetate buffer, pH 4.05, ten parts. (16 ml 0.2M acetic acid + 4 ml 0.2M sodium acetate) 3. Rinse twice in distilled water, 30 seconds to one minute each. 4. Dehydrate quickly in 95% ethanol, 15-30 seconds, then 2 changes of 100% ethanol, one minute each. 5. Clear and coverslip. Source:_Staining Procedures_, 4th edition., George Clark editor. Geoff On 3/28/2012 10:56 AM, Amanda Madden wrote: Good Morning Histonetters! I am looking for advice on my thionin staining recipe and protocol. The recipe I use currently ends up being just under 1% Thionin. It is made by mixing 80ml 1M acetic acid and 14.4ml 1M NAOH to a pH of 4, and then adding 305.6ml 1.3% stock thionin. When I used this protocol last, I rehydrated the mounted rat brain sections (perfused with 4% buffered para) through ETOH to dH20 steps, then left the slides in thionin for 6 minutes, followed by 2x2min rinses in dH20, 1 min in 70% ETOH, 3 min in 95% ETOH, and finally absolute ETOH and xylene for coverslipping. The tissue came out too dark, and what I am wondering is if I should change the thionin recipe to something a bit more dilute, or if I should focus on the baths following thionin, for instance only rinsing in water and moving into a .1% acetic acid in 70% ETOH differentiator? Any help, either by directing my own troubleshooting or even recipes and protocols would be greatly appreciated! Thanks, as always, for being such wonderful resources for those like myself who have such little histology background :) Amanda Madden ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- -- ** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583 mcaul...@umdnj.edu ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] thionin staining
Dear all, I have problems with thionin staining. I am using this on paraffin slices of hypothalami. In the past, I never experienced problems, but the last times, the staining is washed off by going through the ethanol after staining (50%, 70%, 90%, 100%). I tried already a few protocols, but every time the same result. Does anybody have experience with it? regards An Eerdekens ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Thionin staining
An - You didn't mention what cells you were staining, but I assume it's mast cells. Thionin stains are very pH dependent. For mast cells, the pH should be 1. At that pH only mast cells stain. Cheryl Cheryl Crowder, BA, HTL(ASCP) Crowder Histology Consulting 4952 Alvin Dark Ave. Baton Rouge, LA 70820 (225) 772-2865 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] thionin staining
If this is for Nissl staining, the pH of the thionine solution should be 3.5 to 4 and the following rinses should be no more acidic than that. Distilled water is suitable. Alcohol-water mixtures generally remove dyes to a greater extent than 100% alcohol. I shake the washed slides to get rid of most of the water, then go straight to the first of 3 changes of 100% ethanol. Almost no blue is lost from the sections. The first alcohol isn't 100% any more after one or two batches of slides have gone through, so for large numbers of slides it is more economical to go through 95% (quickly) then 3X100%. If you use 50% and 70% alcohol you can expect dye to be extracted from the stained sections. It's not unusual to get unsatisfactory batches of thionine, and for at least one purpose, showing the canaliculi and lacunae of bone, the late Russ Allison found that proper staining could be obtained only with batches of thionine that had been certified by the Biological Stain Commission. See Allison RT (1995) Picro-thionin (Schmorl) staining of bone and other hard tissues. Brit. J. Biomed. Sci. 52: 162-164. The B.S.C.'s tests for thionine are a mast cell stain and a stain for plant tissue infected with a fungus. The dye must also meet spectrophotometric criteria. See Penney DP (2002) Analysis and testing of biological stains - the Biological Stain commission Procedures. Biotech. Histochem. 77:237-275. A minor revision to the criteria for thionine was published in 2008: Lyon HO Kiernan JA (2008) Notes from the Biological Stain Commission. Biotech. Histochem. 83(5):285-288. Make sure your dye really is thionine (CI 52000). There is a dye called thionine blue (CI 52025, Basic blue 25) that is not a substitute. See Conn's Biological Stains or Bryan Llewellyn's StainsFile http://stainsfile.info/StainsFile/dyes/52000.htm . Finally, the word thionine is commonly mis-spelled, without its terminal e. The e should be there because thionine is an amine, in contrast to eosin, which is not. Dictionaries (English or US) give the correct spelling. John Kiernan Anatomy, UWO London, Canada = = = - Original Message - From: An Eerdekens an.eerdek...@med.kuleuven.be Date: Friday, February 4, 2011 3:21 Subject: [Histonet] thionin staining To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Dear all, I have problems with thionin staining. I am using this on paraffin slices of hypothalami. In the past, I never experienced problems, but the last times, the staining is washed off by going through the ethanol after staining (50%, 70%, 90%, 100%). I tried already a few protocols, but every time the same result. Does anybody have experience with it? regards An Eerdekens ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
