Our Safranin O counter stain isn't yielding any color in our stains for articular cartilage. We've increased the concentration of our aqueous Safranin O solution from 0.1 to 0.2 to 0.5 and still are unable to pick up any red coloring. The pHs vary and we were wondering if anyone has any recommendations for a reliable working pH? Thank you.
Dagoberto Pina Jr , apologies: you haven't informed about
i) specimen preparation / processing (i. e. fixation physical=cryo? - chemical=FA? -conditions at the time of withdrawal, further processing - especially un- or de-calcified, dehydration, embedment = paraffin, resin? and finally: sectioning)
ii) which articular cartilage (animal, human, age?)
iii) if you use a certain staining sequence, please, inform about the goal of Safranin O counterstain and the currently performed recipe or staining modification (i.e. what is the stain you use before counterstaining trying to counterstain with SAFRANIN O *)
Thank you in advance,
regards, W.H.M.
*) only as ONE example out of many
cf.: ROSENBERG TC, 1970: Chemical basis for the histologic use of safranin-O in the study of articular cartilage. J Bone Joint Surg 52A: 813-814, 1970
further possibly useful sources :
TRAN et al, 2001: Hematoxylin and Safranin O Staining of Frozen Sections (skin) ,
cf.: Article Hematoxylin and Safranin O Staining of Frozen Sections
= accessible only via PPV(Pay-Per-View) or subscriber @ :https__://onlinelibrary.wiley.com/doi/abs/10.1046/j.1524-4725.2000.09220.x
[NB: before using the wiley-link by Copy & Paste into your browser, delete the twoUNDERSCORE-characters in between https __ and //:....] ,
SCHMUED et al, 1981: Some fluorescent counterstains for Neuroanatomical Studies (cf. p.125 for Safranin O: @
https://pdfs.semanticscholar.org/904e/33eec7fa41d5dbc789e9b6648cb1f42f051c.pdf;
SCHANTZ & SCHECTER 1965: Iron-Hematoxylin and Safranin O As A Polychrome Stain for Epon Sections (Stain Technol, 40, 1965, only via PPV)or subscriber @
https__://www.tandfonline.com/doi/abs/10.3109/10520296509116427) =
Article IronHematoxylin and Safranin O As A Polychrome Stain for Epon Sections
SAWYEK 1940: A Standardized Technic for Safranin O. (Stain Technol, 15, 1940, only via PPV)or subscriber @ https__://www.tandfonline.com/doi/abs/10.3109/10520294009110324?journalCode=ibih19 = https://www.tandfonline.com/doi/abs/10.3109/10520294009110324?journalCode=ibih19
Abstract (copied and pasted only for convenience of reading here):
A method for control of staining with Safranin O is described. The procedure is as follows:
- Overstain the sections, freed of paraffin, 4 hours or more in 0.1% solutions of either light green SF or fast green FCF in 50% alcohol. These solutions are adjusted to pH 2.4 with 0.1 N HCl. Rinse in distilled water.
- Destain at least 30 minutes in Sörensen's Buffer pH 8. Rinse in distilled water.
- Overstain in 0.1 % Safranin 0,4 hours or more. Rinse in distilled water.
- Destain 15 minutes in 0.01 N HCl (pH 2) or in 0.001 N HCl (pH 3) depending on whether light green or fast green, respectively, is the counterstain. The acid solutions are freshly prepared from a stock solution of 0.1 N acid. Rinse in distilled water.
- Dehydrate in two changes of diozan, pass thru xylol and mount in balsam."
Quote from: https://www.bio-world.com/productinfo/2_33_242/3526/Safranin-O-counterstain.html:
Safranin O - Counterstain
" Safranin O is a biological stain commonly used as a counter stain for gram-negative bacteria in the Gram stain procedure. Safranin O has been observed to bind to DNA at a pH 2.21. In the Flemming triple stain procedure Safranin O is used to stain for chromosomes. In late Palaeozoic pteridosperm cuticles Safranin O has shown increased contrast, emphasized cutinization differences, and enhanced the three-dimensional morphology of complex epidermal features when compared with other dyes. The compound has also been used with alcian blue in the histochemical study of the distribution and morphometric characteristics of mast cells. Safranin O has also been used to detect mucin and cartilage. "
Quote from: https://en.wikipedia.org/wiki/Safranin
"Safranin (also Safranin O or basic red 2) is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei red. This is the classic counterstain in both Gram stains, and endospore staining. It can also be used for the detection of cartilage [2],mucin and mast cell granules"
Last remark:
If used as primary stain: Safranin O is a regressive dye and requires destaining and differentiation with picric acid or HCl.
I also face this problem, can’t see any red color at arricular cartilage or epiphyseal plate of rat. I used 10% formalin as fixatives and decalcify in 10% EDTA.
Can you fix this problem or any advice?
Thank you so much
To: Orada Sriwatananukulkit and Dagoberto Pina Jr :
"Currently we aren't picking up....any articular cartilage in our stains".... so my Counterquestion: are you sure about using "articular cartilage" with your study?
"Can you fix this problem or any advice?"
Counterquestion: "is your light microscope color blind?"
All joking apart:
Just to react honestly and with true intention to help:
my former 'requests' {cf. Re [001] i) to iii) } for more detailed information on the matter still persist.
You will not get serious help if you don't include information on your previous processing and staining (sequence, method, dye) the tissue (especially: unfixed-prefixed cryo- / or paraffin / resin embedding). Trouble shooting still isn't a 'digitized' multiple choice test!.
It is not possible (at least not for me) to 'retrieve' or going to fetch [like a dog] "a stick" which turns out to have been thrown only fictitiously....
QUOTE: from Hyllested et al, 2002:
(regarding articular cartilage histochemistry)
" .... Time of fixation, pH, temperature, osmolarity, fixative penetration (diffusion) of tissue as well as volume, buffering and freshness of liquid solutions should always be considered, since these factors are important for efficacy....(NB: added by me: of staining too).
For me it is interesting that RG-requests on difficult and very specific (scientific, methodological, technical....) matters frankly are placed,
but aren't - obviously - kept on "familiar terms", i. e. answers either aren't read and / or- despite announced further enquiry for more detailed information on processing parameters aren't appropriately answered.
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