Author Topic: Help interpreting caspase and Ki67 stain  (Read 5357 times)

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Offline ic2133

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Help interpreting caspase and Ki67 stain
« on: November 08, 2012, 11:34:21 AM »
Hello,

We had hair follicles sent out to a third party for H&E, caspase, and Ki67 staining. I have no experience with these stains and need some help interpreting the results. I have copied and pasted the protocol used for each stain below.

My questions are:

-If the Caspase and Ki67 slides were counterstained by dipping only in hemotoxylin (blue), why are there red dots on the slides?
-Is brown color on the caspase and ki67 slides the signal for those proteins? I ask this because the H&E sildes also have brown color. I'm wondering if it's just the color of the hair follicle itself or if the H&E stain results in that color? I don't have the protocol for the H&E stain the guy used since I guess it's a common one.
-I'm also confused with the naming of the control slides. How can I interpret those? The (-)(-) control seems to have no brown at all but it's also possible that he sectioned way past the follicle itself.

I would ask the guy himself but I was told not to ask him questions about interpretation...

Any feedback would be great. Thank you in advance!
-Irene





Procedure for Ki-67 IHC

   5 micron tissue sections deparaffinized and hydrated to water.
   Antigen retrieval for 1hour @ 90 degrees C in Trilogy solution (Cell Marque #92p-10,)
   Slides rinsed for 10 minutes in TRIS buffered saline and Tween (Thermo Scientific # TA-999-TT)
   Protein block 10 min ( BIOCARE MEDICAL #BS966M,)
   Rinse in Tris buffer 5 min
   Primary ab incubation (Ki-67) 1 hour @room temperature
   Rinse in Tris buffer 5min
   Alk Phos Probe 20 min (BIOCARE MEDICAL #UP536L)
   Rinse in Tris buffer 5 min
   Alk Phos Polymer 30 min (BIOCARE MEDICAL # MRAP536L).
   Rinse in Tris buffer 5 min
   Chromogen Red 15 min (BIOCARE MEDICAL # FR805CHC)
   Rinse in water and counterstain with 2 dips in Hematoxylin.
   Dehydrate in graded alcohols and clear in xylene and coverslip.

Procedure for Caspase IHC

   5 micron tissue sections deparaffinized and hydrated to water.
   Antigen retrieval for 1hour @ 90 degrees C in Trilogy solution (Cell Marque #92p-10,)
   Slides rinsed for 10 minutes in TRIS buffered saline and Tween (Thermo Scientific # TA-999-TT)
   Protein block 10 min ( BIOCARE MEDICAL #BS966M,)
   Rinse in Tris buffer 5 min
   Primary ab incubation (Cleaved Caspase3)  1 hour @room temperature
   Rinse in Tris buffer 5min
   Alk Phos Polymer 30 min (BIOCARE MEDICAL # MRAP536L).
   Rinse in Tris buffer 5 min
   Chromogen Red 15 min (BIOCARE MEDICAL # FR805CHC)
   Rinse in water and counterstain with 2 dips in Hematoxylin.
   Dehydrate in graded alcohols and clear in xylene and coverslip.



Help interpreting caspase and Ki67 stain
« on: November 08, 2012, 11:34:21 AM »

Offline gula

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Re: Help interpreting caspase and Ki67 stain
« Reply #1 on: November 08, 2012, 11:47:21 AM »

Melanin is the naturally brown pigment in hair. So you can't differentiate between DAB and Melanin, if you are unlucky.
It would be the best to stain with red chromogen, when dealing with skin and hair. But red chromogen often tends to leak out and isn't as clear as DAB.
Onother method is to bleach the tissue (0,25% potassiumpermangante 1 hour and 4% Oxalacid 5 min). This method is stated as suitable for IHC - personally not tested!

In your slides the IHC-result is shown with red chromogen: red dots are the IHC-result, should be the nuclei in KI67 staining. For Caspase staining look into the datasheet of the antibody to learn.

What a person, that can't be asked about his staining and naming??
The  (-)(-) looks different than the others. It may be a tissue, that doesnt express these proteins. The (-) looks like hairfollicel with IHC without primary antibody, if the colour is brown (=Melanin). The red staining of the negative control (=background) has to be related to your specific red staining. all red minus background=specific
The positiv control is positiv, but what for?

hope this helps
gula
« Last Edit: November 08, 2012, 11:59:39 AM by gula »

Offline NexusIHC

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Re: Help interpreting caspase and Ki67 stain
« Reply #2 on: December 11, 2012, 01:43:07 PM »
It does appear that the brown coloring is the result of melanin from the hair follicle.

I agree with gula that the (-)(-) slides should be some sort of tissue devoid of nuclear proliferation and (-) should be a positive control tissue without the primary antibody.


Offline MT Scientist

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Re: Help interpreting caspase and Ki67 stain
« Reply #3 on: January 03, 2013, 12:25:29 PM »
Judging by the pictures and the protocol you posted, the red color is the POSITIVE signal for the antibody(s) of interest [Chromogen Red as the substrate for Alk Phos]. 

The source of brown color in your (-) control slide is unclear (as it is not present in the -- slide), but your (-)(-) slide appears to be a different tissue from that used in the (-) and (+) slides.

Re: Help interpreting caspase and Ki67 stain
« Reply #3 on: January 03, 2013, 12:25:29 PM »