Good day, everybody. I have stained sagranin O rats bones with an intra-articular fracture. In all figures in papers it should be stained blue (bones) and red (cartillage). But as it seen at the figures bone structures stained pink-violet.
Protocol is so:
1. Deparafinize and rehydrate.
2. Weigert's Iron Hematoxylin stain for 10 min.
3. Wash in tap water for 10 minutes.
4. Fast green stain for 10 minutes.
5. Wash fast in 1% acetic acid for 10 seconds.
6. Stain Safranin O for 5 minutes.
7. Dehydrate and clear in xylene.
May be problem in protocol? May be somebody can recommend another protocol?
La safranina en combinación con el azul de alciano se emplean en la diafanización a claramiento de Dowson para embriones o fetos. Quizá las fotomicrogrfías que ha visto se tomaron de esta técnica
German Isauro Garrido-Fariña ,Sorry, I am not fluent in the Spanish. Can you write the protocol with alcian blue?
English translation: „Safranin in combination with Alcian blue are used in crosstalk to Dowson's clearance for embryos or fetuses.
perhaps the photomicrographs you have seen were taken from this technique“
You might search google for
Search phrase (s):
„dowson clearance alcian blue“,
„enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage“ etc… shall come back later , regards WHM
(==> one out of many hits: https://www.researchgate.net/figure/Alcian-blue-and-Alizarin-red-staining-of-a-E145-and-b-E165-embryos-Images-were_fig1_259989719
CAVE: LONG post!
Dmitry Zinovkin ,
as promised, back to this your Request, just to clear up:
First of all, I’d propose you correct – at least - the title of your Query in RG to:
>> Technical problems with Safranin O stain. Should it be so stained? SAFRANIN O stainChahles H. SAWYEK< by the reading and copy/paste algorithm of publisher (cf: https://www.tandfonline.com/doi/abs/10.3109/10520294009110324?journalCode=ibih19)
A Standardized Technic for Safranin O, Stain Technology, 15:1, 3-7, DOI: 10.3109/10520294009110324, which in the SUMMARY reads:
>>ABSTRACT.-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 distiiled water.
- Destain 15 minutes in 0.01 N HCl (pH 2) or in 0.001 N HC1 (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 dioxan, pass thru xylol and mount in balsam.
Is it only safranin O you used or you counter stained with other stain? From the above photomicrograph you did not do differentiation very well. Your differentiation was poorly done. I am in to Plant Systematics (Taxonomy and Anatomy). We used series of grades of ethanol to carry out differentiation. If it is also apply in animal histology you can try it also. Thanks.
Akinloye A. J. PhD.
Thank you all for your answers, I found the problem. The intence of the blue color depends from the method of dacalcination. I used EDTA. In case of formic or HCl acid decalcination bones gives intence blue color.
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