Author Topic: elastin staining  (Read 13085 times)

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Offline REDDY

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elastin staining
« on: July 28, 2005, 01:10:50 PM »
Hi,
  i'm trying to do elastin staining protocol that was given in this website.I have did , but couldn't see any elastin fibres.Does it mean i should put in antigen retrieval for more time ,even if i counter stain with gill 3 hematoxylin the sections are all blue color couldn't make out elastin and without counterstain sections are looking brown color but couldn't see the elastin fibres.can you please suggest me ...
Thanks
reddy
-Vasanthi Reddy

elastin staining
« on: July 28, 2005, 01:10:50 PM »

Offline richard03

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elastin staining
« Reply #1 on: July 28, 2005, 06:32:29 PM »
Are you using Clone BA-4 from Novocastra? Did you follow this protocol?

http://www.ihcworld.com/_protocols/antibody_protocols/elastin_novocastra.htm

I have used this protocol with good success. Here are some images stained with BA-4 antibody: http://www.ihcworld.com/imagegallery/thumbnails.php?album=search&type=full&search=elastin

You must use Proteinase K digestion for pretreatment. Other antigen retrieval method may not work well. Also this antibody does not work on acetone fixed frozen sections. Were you using paraffin sections?

Richard

Offline REDDY

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elastin staining
« Reply #2 on: July 28, 2005, 06:43:56 PM »
THNX RICHARD, YEA I WAS USING SAME PROTOCOL AND ANTIGEN RETRIEVAL FOR PARAFFIN SECTIONS.
 YES I'M USING SAME CLONE FROM NOVOCASTRA.

i'M DOING SAME AS THE PROTOCOL SAYING.I THOUGHT MAY BE THE ANTIGEN HIDDEN , SO I HAVE INCREASED TIMING OF ANTIGEN RETRIEVAL (PROTEINASE K) ABOUT 1 HR.THE RESULTS ARE YET TO COME.DID YOU TRY DOING WITH HUMAN SKIN ????
 
 SEND ME SOME SUGGESTIONS
THNX
REDDY
-Vasanthi Reddy

Offline richard03

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elastin staining
« Reply #3 on: July 28, 2005, 07:06:55 PM »
Yes, I have done with human skin and it works well.

Did you get any staining even weak staining or NO staining at all?

I think the key step is proteinase K digestion. You may use some other tissues such as kidney as positive control and give it a try.

Richard

Offline REDDY

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elastin staining
« Reply #4 on: July 28, 2005, 07:11:42 PM »
RICHARD,
   i have got gill3 hematoxylin(counterstain) for about 1min and 5 sec all over the tissue but couldn't able to see the elastin fibres.And i did without counterstain still not able to see but the sections are looking brown at this time.First thing i thought was antigen so i increased the timing to 1 hr.wat you think about DAB ?
  when i compare with my images and ur images ...your images looks great and the hematoxylin was light.did you do with diluted hematoxylin?

Thnx
reddy
-Vasanthi Reddy

Offline richard03

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elastin staining
« Reply #5 on: July 28, 2005, 07:24:12 PM »
I guess you still got staining but just weak. So in this case counterstain is more important. You definitely do not want to use Gill3 concentrated. You must dilute 1:5 even 1:10 or more. And also only stain 10-30 seconds and no more.

By the way, what DAB are you using?

Hope this helps and good luck.

Richard

Offline REDDY

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elastin staining
« Reply #6 on: July 28, 2005, 07:41:09 PM »
DAB from vwr .i'm using DAB Peroxidase Substrate Solution Black that was given in the protocol.
thnx
reddy
-Vasanthi Reddy

Offline richard03

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elastin staining
« Reply #7 on: July 28, 2005, 11:07:17 PM »
If DAB-Black was used, it will be better to use nuclear fast red for counterstaining. Otherwise black and blue are difficult to distinguish.

Richard

Offline excalibur

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elastin staining
« Reply #8 on: July 29, 2005, 10:58:55 AM »
Reddy, have you tried just a plain Verhoff-Van Geison or Orcein for elastic

Artery cross-sections make wonderful controls for elastin.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline REDDY

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elastin staining
« Reply #9 on: July 29, 2005, 12:28:14 PM »
YEA WE HAVE DONE EVG STAINS BUT WANT TO TRY WITH IMMUNOSTAINS........

THNX RICHARD

REDDY
-Vasanthi Reddy

Offline REDDY

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elastin staining
« Reply #10 on: July 29, 2005, 12:43:22 PM »
RICHARD,
  CAN YOU PLEASE SEND SOME IMAGES OF NUCLEAR FAST RED OF ELASTIN/ANY ANTIGEN IMMUNOSTAINING

THNX
REDDY
-Vasanthi Reddy

Offline richard03

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elastin staining
« Reply #11 on: July 29, 2005, 03:03:34 PM »
Hi Reddy,

Nuclear fast red is for counterstain but not for elastin. When you use DAB-black or blue then nuclear fast red should be used for counterstain instead of hematoxylin. Here is an image for your reference.

http://www.ihcworld.com/imagegallery/displayimage.php?album=1&pos=118

Richard

Offline REDDY

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elastin staining
« Reply #12 on: July 29, 2005, 03:33:39 PM »
hi richard
when i increased th etime of antigen retrieval from 20 mts to 1 hr.....showing some elastin fibres but those are light brown color.So i thought of doing more time or increasing the time of incubation DAB from 10mts to 30 mts.May be i will get somewhat light black colored elastin fibres.

Thanks a lot
Reddy
-Vasanthi Reddy

Offline richard03

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elastin staining
« Reply #13 on: July 29, 2005, 04:12:05 PM »
10 min for DAB is enough. I would increase the antibody concentration or incubation time or both instead.

Richard

Offline REDDY

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elastin staining
« Reply #14 on: August 02, 2005, 04:18:48 PM »
HI ,
  WHEN I INCREASED THE TIME OF INCUBATION FOR DAB TO 30MTS ,THE ELASTIN LOOKING CLEAR AND BLACK FIBRES.I DID TRY DOING WITHOUT COUNTERSTAINING.
THANKS
REDDY
-Vasanthi Reddy

elastin staining
« Reply #14 on: August 02, 2005, 04:18:48 PM »