Stab Wound Injury of the Zebrafish Adult Telencephalon

[John C Calhoun]

Hi, I have changed my experiment and I'd like to explain it then ask my question.

As suggested in the title, I will be delivering a stab wound to the telencephalon of an adult Zebrafish and allowing it to regenerate for a couple days then sacrifice the fish, dissect the brain, embed it in paraffin, slice it with a microtome, and perform immunohistochemistry on the slices. I am following a protocol on Jove (The Journal of Visualized Experiments) and changing something in the experiment as a variable (I have not yet figured it out). I understand how to do everything except the immunohistochemistry and I would like to ask for the clarification.

Here's the section I have some confusion on:

1. Block non-specific binding sites for 1 hr at RT in blocking buffer (0.1% (v/v) 10% Tween 20, 0.2% (w/v) BSA, 1% (v/v) DMSO in PBS).
2. Remove the supernatant and add 250 μl of antibody diluted in blocking buffer overnight at 4 °C. Alternatively, incubate the primary antibody for 2 hr at RT. Then wash 3x for 10 min in PTW. Note: For PCNA staining use a 1:400 dilution (mouse anti-PCNA. For details see table of materials). Multiple primary antibodies can be incubated at the same time.
3. Incubate the sections in secondary antibody for 2 hr at RT then wash 3x for 10 min in PTW. Note: 1:1,000 dilution in PTW, for detail see table of materials. Multiple secondary antibodies can be incubated at the same time.

How do I dilute the antibody??

[deleted-140482]

Hi John,

This sounds like a very sophisticated project. I assume you have access to a lab (university level at a minimum) that has a microtome and some experience in embedding and cutting tissues, as well as immunohistochemistry. If so, then I'm sure the members of that lab can help you with the details of your staining, but of course, it's great to understand things as much as possible in advance. I'm not sure exactly what the problem you are having is with diluting the primary antibody, though. In immunohistochemistry we look at the levels/location of specific proteins using an antibody specific to the protein of interest. You don't specify what protein you think you'll be staining for, but you'll need to find an antibody specific to that protein (this will be your primary antibody). You can usually buy this if the lab you are working in doesn't already have it, but they are not cheap (often ~$300). You'll only get a small amount of very concentrated antibody, so you'll want to dilute it for use in the staining. Oftentimes the antibody will give you instructions for how much to dilute it by, but often a good starting dilution is about 1:10,000. As your protocol says, you'll usually dilute your antibody in the same buffer you used for blocking, so you might make up 10mL of antibody by combining 10mL of blocking buffer with 1uL of primary antibody. Then you would add the primary antibody to each of your slides and incubate. You would do something very similar with your secondary antibody, but often at a different dilution.

I hope this helped answer your question. If not, feel free to post again in this thread, and remember, the more information you give us about your project, the more help we can be.

Good luck!
JMP

[John C Calhoun]

As far as I can tell, I am not to be looking for a protein but to analyze the entire hemisphere. I appreciate your help and ask that you take a look at the protocol here: http://www.jove.com/video/51753/stab-wo ... nvestigate

The section "Representative Results" seems to cement my assertion.

[caraskl]

Hi,

I reviewed your article, and the purpose of your experiment is to observe the cellular and molecular effects of mechanical injury on brain tissue using techniques such in siti hybridization and immunochemistry. When I performed immunochemistry in college, we used primary antibodies to bind the antigen, and we used secondary antibodies to make the primary antibody/antigen complex visible. A similar procuedure might be at work in your experiment. In your exaperiment, anti-PCNA antibodies would bind PCNA antigens in the injured hemisphere of the brain. PCNA is the most sensitive marker of brain injury, and it is often upregulated during injury. Both PCNA and SLOOB stainign would indicate radial glial cells. In addition, anti-GFP antibody would detect OPCS near the lesion. Only one hemisphere of the brain should be injured, and thus antibody staining patterns should be different for the injured and uninjured hemisphere. Thus, you can assess how injury induces molecular and cellular changes in the brain.

Katherine Caras

[John C Calhoun]

If you could tell me how much PCNA I need and how to get it at a 1:400 concentration, I would be forever thankful. I am only doing immuno not in situ and can probably only afford the PCNA.

[deleted-132180]

Hi there,

The amount of anti-PCNA antibody you need depends on how many sections you're going to be staining. Since it seems like you have access to a lab space to do all of this work, does your lab happen to have anti-PCNA antibodies? If yes, that's great. If not, you would have to buy some, and according to the protocol you sent, this is the one you want to look for (http://www.dako.com/us/ar38/p105790/pro ... %20country). In addition, the protocol says to stain individual sections each in a well of a 24 well plate, and in each plate, they want you to add 250 microliters of antibody solution. Having the anti-PCNA antibody at a 1:400 concentration means that you want to add 1 microliter of your anti-PCNA antibody into 400 microliters of blocking buffer. That means, for 250 microliters of blocking buffer, you want to add 0.625 microliters of antibody to that (take 250 and divide it by 400). However, if you're staining all of your brain sections with the same primary antibody solution, you can just make the entire batch together (for example, if you have 4 sections to stain, that means 1000 microliters of primary antibody solution total, which means 2.5 microliters of anti-PCNA antibody into 1000 microliters of blocking buffer, and after mixing that up, you just add 250 microliters of this mixture to each of your sections).

Hope that makes sense. Let us know if you have anymore questions.

Connie

[John C Calhoun]

Complete success! I was able to do it at home too, no need for a uni lab.