Methods and Techniques Discussion > Immunohistochemistry (IHC)

Issue with RIHC of cryofrozen tissues

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kappadonna:
Hello IHCWorld,

I am trying to stain frozen sections of lactating mouse mammary glands with a beta casein (milk protein) antibody from Santa Cruz.  I am also using frozen normal mouse kidney sections as a negative control.  Each time I carry out my IHC protocol (which is actually a rapid staining protocol so I can then proceed to laser capture), all of my tissues end up with a brown background after I dehydrate them with ethanol and xylene.  My positive control tissue appears to show that the primary antibody is working but the background here AND the background in the negative control samples (ie. secondary only treated kidney and mammary tissues) is driving me up the wall.  I have a notion that the background may be the result of the dehydration process at the end of the protocol.  I say this because I have tried visualizing the tissue under the microscope during each step of the protocol and noticed that there was no background observed following application of the DAB and hematoxylin steps.  Does anyone know what gives?  Does anyone know if tissue dehydration process may be responsible for this?

Thanks muchly!  And below is some other pertinent information regarding my protocol.

The matrix I set up for positive and negative control tissue is as follows:
Mouse Mammary - one sample stained with primary+secondary; one sample stained with secondary only
Mouse Kidney - one sample stained with primary+secondary; one sample stained with secondary only

I am using the Dako EnVision+ System-HRP (DAB).  The antibodies I am using are anti-mouse beta-casein (M-14; sc17971) goat pAb (1:50-1:100 dilution) and donkey anti-goat IgG-HRP (sc2020) (1:500-1:1000 dilution).

Here is the protocol I am using for these frozen sections:
1. After slicing tissues at 4 microns, they are mounted and fixed in cold acetone for 30 sec
2. Slides are washed for 3x 30sec in PBS
3. Peroxidase block with 1% H2O2 in PBS for 5min
4. Wash for 3x 30sec in PBS
5. Block with 1.5% horse serum in PBS for 5min
6. Apply primary antibody in 1.5% horse serum/PBS for 10min
7. Wash for 3x 30sec in PBS
8. Apply secondary antibody in 1.5% horse serum/PBS for 10min
9. Wash for 3x 30sec in PBS
10. Apply DAB+ Substrate-Chromogen for 5min
11. Wash for 3x 30sec in ddH2O
12. Counterstain with hematoxylin
13. Wash immediately with several changes of dH2O
14. Dehydrate tissue by dipping in each buffer for 10sec each: 95% ethanol, 95% ethanol, 100% ethanol, 100% ethanol, xylene 1, xylene 2, xylene 3
15. Allow samples for air dry before inspecting under microscope

gula:
You may compare the results and find "the point of background", when you use a water-miscible mounting medium without the need of dehydration.
Try a negative control without any antibody, just pretreatment and hematoxylin. If the slide is clean add one step from the end of the protocol. (1. DAB, 2. sec. AB)
Try to let the slides airdry without dehydration. It takes longer, but should have the same effect.
I've never experienced background due to dehydration, but my slides are usually coverslipped. It may be deposits that cristallize when dry and therefore are only seen after dehydration.
gula

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