ki 67 Staining

[Leonard Cheung]

I have stained my rat sketelat ischaemic muscle w/ mouse anti-ki67 rat MIB-5 (Dako) as following:

-10mM Citratic Buffer pH6 for 5 mins high power 2 times
-Primary Ab dilutons of 1:25, 50 for 1hr 30 mins at Rm temp.
-Dako EnVision DAB kit: peroxidase block 15 mins, secondary Ab 30 mins at Rm temp., 1:50 DAB for 1 min 30 sec.
-Counterstain with Hematoxylin for 15 sec

However, the backgrounds of two slides are a little bit high, but the positive intensity are low. Also, some areas with high background and some with low in same tissue section. Why? How can I improve the result? Thanks

[ole]

Hi
You could try increasing your retrieval time in citrate buffer, or change to the more efficiant TE-retrieval-buffer. If this does not help, you could try changing detection-system.

The envisionsystem have rather large backbones/large polymers and a efficient retrieval is often necessary for achieving optimal staining in some of the nuclear antigens using these detection-systems.

mwo should work, even tho i find eg pressure cooker to be better than mwo concerning Ki67.
Mwo; i guess you have different power levels? I would suggest bringing buffer to boil (high effect), then low effect (simmer) for the appropriate time (avoids most of the evaporating "problem")

Ole

[richard03]

Are you using capillary gap slides for the staining? If so, it often produces uneven staining (bottom stained stronger than top). To solve this problem I often reduce primary antibody concentration. You may try 1:100 or more diluted antibody.

As Ole suggested, increasing antigen retrieval time may help as well.

Richard

[Leonard Cheung]

Thx Richard & Ole,

Ole, I'm sorry that I don't understand the meaning of "large backbones/large polymers" Would you explain it? 1mM EDTA pH8 was also used for antigen retrieval, also high power for twice 5 mins, but the background was very noisy. is it similar to TE buffer? Thanks.

Richard, the Poly-S slides were used, if so, are reducing the Ab concentration and longer the retrieval time still useful? Also, what is capillary gap slides? Thanks.

Leonard

[richard03]

I think it will be still useful for reducing Ab conc. and longer retrieval.

When using Capillary Gap slides, they usually stand vertically so the bottom of sections get more staining and the top of sections get less intense staining.

Richard

Thx Richard & Ole,

Richard, the Poly-S slides were used, if so, are reducing the Ab concentration and longer the retrieval time still useful? Also, what is capillary gap slides? Thanks.

Leonard

[ole]

Leonard
I have not read the article myself but i guess there are some info about it in this article??
Shi S-R.... Sensitivity and detection effiency....., Applied imm and mol morp  7:201-   (1999)

The envision+ systems contains rather big molecules so one might get steric and "penetration" problems with these big molecules especially on some of the nuclear antigens.
So one might need a more "optimal" retrieval if its sterical problem you are encountering, can explain your low signal?.
I have had my share of problems with different Ki67 antibodies. I got unconsistant staining back in 1998-1999. I had to increase the citrate buffer retrieval 2-3x or use the TE-buffer to overcome the problem. I used EDTAbuffer in 1998-1999 but changed to TE buffer in 2000. EDTA and TE performs about the same but with the envision system i found TE-buffer to be slighly better eg on several nuclear antigens.

If you have one, try a link between antibody and the envision system, might do the trick :wink: . Perhaps rabbit-anti-mouse followed by envision-anti-rabbit??

Ole

[ImmunoNYC]

I have a protocol for this antibody which I can look up when I get to work if you like.

Importantly, I would *not* use the Envision mouse kit for rat tissue. Rat and mouse IgGs are very similar and the anti-mouse secondary is likely picking up endogenous Rat IgG in the tissue. I would use a donkey anti-mouse adsorbed against rat instead. How does your negative control look? What are you using as positive control?

Also what do you mean you use DAB 1:50??? I don't get that part! Isn't the EnVision kit one drop of chromagen in 1ml of substrate buffer???

Good luck!

I have stained my rat sketelat ischaemic muscle w/ mouse anti-ki67 rat MIB-5 (Dako) as following:

-10mM Citratic Buffer pH6 for 5 mins high power 2 times
-Primary Ab dilutons of 1:25, 50 for 1hr 30 mins at Rm temp.
-Dako EnVision DAB kit: peroxidase block 15 mins, secondary Ab 30 mins at Rm temp., 1:50 DAB for 1 min 30 sec.
-Counterstain with Hematoxylin for 15 sec

However, the backgrounds of two slides are a little bit high, but the positive intensity are low. Also, some areas with high background and some with low in same tissue section. Why? How can I improve the result? Thanks