Products and Equipments Discussion > Histology Products

NovaUltra Safranin O Stain Kit

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NKouris:
I purchased the Safranin O  Stain Kit (http://www.ihcworld.com/catalog/p4159/NovaUltra-Safranin-O-Stain-Kit/product_info.html), and ran control and chondrogenic samples.  I had fixed my samples in 4% PFA, dehydrated and paraffin embedded, followed by sectioning and then the staining protocol according to the protocol provided with the kit (Weigert's Iron Hematoxylin for 10min, Fast Green for 5min, Acetic Acid for 10sec, and Safranin O for 5min).  All of the samples are "positively" stained, that is they have red staining with no green/teal background staining. 

I have had samples from the same batch previously stained by a 3rd party professional and the controls were bright blue/teal and the chondrogenic samples were red (as expected). 

I am trying to troubleshoot why all the samples are red when I ran the samples with this kit and protocol.  Does the Safranin O incubation time of 5min need to be reduced to avoid this overstaining false positives?



gula:
Was there a second rinsing step after the Safanin O? You can try to rinse it in the 1%acetic acid afterwards and prolong it till the safranin O is sweapt out of the unwanted areas. If acetic acid doesn't bring the success, prolong rinsing in 96% ethanol. One of these should bring a differentiation of the staining.
gula

NKouris:
Thank you for the advice gula.  I have been in contact with Richard and he informed me I was using the old protocol, with this order:
Iron Hematoxylin
tap H20
Fast Green
Acetic Acid
dH2O
Safranin O

I have now used the updated protocol on your website with this order:
Iron Hematoxylin
tap H2O
Safranin O
dH2O
Fast Green
Acetic Acid

This has helped reduce the overwhelming red stain in all samples, but there is little to no blue/green background stain.  This is true even with a reduction of Safranin O to 2min, and an increase in dehydration of 95% EtOH for 5min.  To get the vibrant background stain should more time be spend on the Fast Green step >10min?

I have sent images to Richard to better illustrate the problem.  Any additional help is greatly appreciated.

gula:
Hi,
I'm also just a user of ihc-world and have no responsibilities : ).
Now in this conversed order. Rinsing after Fast Green should be minimized in acetic acid and diluted ethanols. Perhaps prolonged rinsing after Safranin O and before Fast Green, makes the stain more clear.
good luck
gula

NKouris:

--- Quote from: gula on November 07, 2012, 01:51:02 PM ---Hi,
I'm also just a user of ihc-world and have no responsibilities : ).
Now in this conversed order. Rinsing after Fast Green should be minimized in acetic acid and diluted ethanols. Perhaps prolonged rinsing after Safranin O and before Fast Green, makes the stain more clear.
good luck
gula

--- End quote ---

Oh, I see.  Then I really appreciate your feedback!  I increased the Iron Hematoxylin and Fast Green steps by 2-fold to 20min, and reduced the acetic acid step by half to 5sec and now I have decent blue background staining.  Not vibrant as I have seen by others, but there.  This has reduced the safranin O staining as well, so I may try increasing the Fast Green to 30min to become more vibrant and increase the Safranin from 2 to 3min to regain some of the red stain.  Thanks for all the help, on track here!

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