Re: [Histonet] breast processing
Alcoholic Formalin or Penfix in the processor helps some, if the tissue
is grossed in at a reasonable thickness..
Featherstone, Annette wrote:
>Does anyone have any suggestions for better tissue processing for fatty tissue such as breast? We are experiencing insufficent dehydration, clearing and infiltration.
>Annette Featherstone HT/MLT
>[mailto:firstname.lastname@example.org]On Behalf Of
>Sent: Sunday, April 02, 2006 13:01
>Subject: Histonet Digest, Vol 29, Issue 2
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> 1. My frustrating experience with double fluorescence staining!
> (Chengming Wang)
>Date: Sat, 01 Apr 2006 23:25:00 -0600
>From: "Chengming Wang"
>Subject: [Histonet] My frustrating experience with double fluorescence
>Content-Type: text/plain; charset=US-ASCII
>I am new with histology, and posted yesterday a message of: Help
>needed!!! Immunofluorescence double staining with mouse lung. Thanks a
>lot for the several helpful responses. As asked, I am here putting on
>my concise protocols for your check:
>Sampling: Cut a small cut along the mouse trachea, injected neg 50
>(similar to OCT). Then quick freeze with liquid nitrogen, and the stored
>@ -80; After section with 6 um, slides were fixed with icy acetone for
>5 minutes. Quick dry, and then stored @ -20 till use.
>Staining: 1. Washing 3 X 5 mins; Blocking Buffer (10% Rabbit serum; 5%
>BSA; 0.1% Tween 20) for 1 hour at room temperature;
> 2. Tapping away the blocking buffer, and applied first
>antibody for 1 hour; then wash 3 x 5 min'
> 3. 2nd antibody 1 hour; washing 3 x 5min;
> 4. Mounting media, read slides. Count stained with
> 5. For double staining, I did the same thing, except
>using mixed first antibodies and mixed second conjugated antibodies.
>Designing: Stain 1: Rabbit anti-mouse antibody, and donkey anti-rabbit
>conjugated with Alexa Fluor 488;
> Stain 2: Rat anti-mouse antibody, and donkey anti-rat
>conjugated Alexa fluor 594;
>My problems: The individual staining signals are good. But there is
>some match color between two channels. For example, I could see weak and
>similar green signal when I applied Alexa 594. Basically, I could see
>the weak but true alveoli structure of mouse
> Then I performed double staining, I could see
>lots of signal match between red and green channels, which should not be
>true. In this way, I could see basic alveoli structure of mouse
> lung in both green and red channels. By the way,
>I tried to red the slides before staining and after blocking, the
>autofluorescence is very very weak.
>I am really frustrating with this situations, and please feel free to
>tell me how I could figure out this problem. Any of your suggestions and
>comments are very much appreciated.
>DVM, M.S., Ph.D.
>Department of Pathobiology
>College of Veterinary Medicine
>264 Greene Hall
>Auburn, AL 36849-5519
>Voice: (334) 844-2624
>Histonet mailing list
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