Can anyone walk me through IHC/IF Staining of Cryosectioned Brain Tissues?

1- You can use positively charged slides for better attachment. Or you can coat your slides with agents like gelatin or lysin. Both protocols are quite easy to find online and easy to oparate. Also make sure your slides are completely dried before you start.

2- Yes you should perform antigen retrieval since you use a formaldehyde based fixation. For that, you can use enzymes or you can boil your sections in buffer solutions like citrate buffer or tris-EDTA buffer.

I mostly prefer using citrate buffer pH:6.0. Prepare the buffer, put your slides in a challet filled with buffer solution and microvawe for 5 mins (for three times). Let your slides cool down a bit between microvawe sessions.

But if you have some problems with adhesion, you may prefer trypsin for retrieval. You can find the protocol for that in the link: http://www.ihcworld.com/_protocols/epitope_retrieval/trypsin.htm#:~:text=The%20trypsin%20based%20solution%20is,enhancing%20staining%20intensity%20of%20antibodies.

Note: Since you're working on frozen sections skip first two steps of the protocol. The antigen retrieval step is the 4th step alone.

3- Hydrophobic pen+humidity chamber works fine for me. Mark your sections, drop antibody solutions, place a plastic coverslip onto the section, make sure there's no bubbles on the slide and incubate them in humidity chamber at +4C overnight. Don't worry, if you place coverslip w/o bubbles your sections wont dry out. I use very small amounts of antibody solutions (like 20-30 microliters) and it's working without any problems.

4- I believe if you use parafilm instead of h.phobic pen, the parafilm may melt down in antigen retrieval step. If you don't have a h.phobic pen, you can use a diamond pen or some sharp equipment to scratch the surroundings of the section.

Anton Lennikov

Use pap pen hydrophobic barrier to retain liquid on top of the sample and incubate in a humidity chamber to prevent evaporation or use thin parafilm strips to form a rectangular shape around the sample. Parafilm takes more effort and not recommended if a hydrophobic pap pen is available.

Add 0.1% Triton X100 10-15 minutes for permeabilization

Remove 0.1% Triton X100

Wash cells with PBS.

Add 2.5% Bovine serum albumin (BSA) and block for 1 hour at RT Instead, 1 hour at room temperature. Blocking can be done overnight at 4C incubate in a humidity chamber to prevent evaporation.

If the primary antibody is frozen aliquot melt it on ice and spins down the antibody vials.

Dilute primary antibody or mixture of primary antibody in 2.5% BSA. Make sure to properly vortex the solution. The standard concentration of the primary antibody is 1:100-1:250. Check the datasheet for details.

Remove the BSA and add the primary antibody or mixture of primary antibodies for multichannel staining in 2.5% (BSA). Leave the primary antibody overnight in the humidity chamber at 4C. Do not incubate your primary antibody longer than 24 hours. i.e., adding primary on Friday and resuming staining on Monday will produce a noisy image due to the excessive binding. In general, you cannot fix this by extensive washing. Always leave one sample in 5% BSA without primary antibody it will be later used to null your exposure time to the non-specific background (null sample).

Collect the primary antibody as they can be reused multiple times within the span of up to 3-4 weeks if stored at 4 degrees. Antibodies are expensive, but Eppendorf tubes cost a few cents. It can save a lot of time and resources if you do the same staining with multiple samples.

Prepare slide washing rack and fill it with 0.02% Tween 20 in PBS (PBS-T) or TBST with 0.02% Tween.

Wash the slides by keeping the slides in the washing buffer on a shaker at RT for 5-10 min x 3 times.

Wash away PBS-T with PBS.

Renew pap pen barriers if they were washed off during washing.

Prepare secondary fluorescent chromophore conjugated antibody against corresponding host species of primary antibody. i.e., if your primary host is a rabbit use anti-rabbit Cy3 or anti-rabbit Cy5 (note you can’t see 647 (Cy5) fluorescence with your eyes only camera, as its emission is in infrared). If you are conducting multichannel staining, you can use a mixture of secondary antibodies. The standard concentration of secondary antibody 1:1000 adds DAPI for nuclear counterstaining 1:1000 of 1 mg/ml DAPI stock. Properly vortex the solution. Add secondary antibody to all samples, including the null sample.

Incubate in the humidity chamber for 1 hour at RT, protect from the light from this point.

Wash the slides by keeping the slides in the washing buffer on a shaker at RT for 5-10 min x 3 times.

Cover the samples with PBS to remove traces of Tween-20, as Tween-20 is slightly autofluorescent.

Drop off the excess liquid and cover the samples with antifade reagent and mount with a cover glass.

Use the xylene-based mounting media or nail polish to secure the edges of the cover glass to the sample glass.

Observe the samples with a fluorescent or confocal microscope within 7-14 days after staining. Samples can be stored at 4C in the dark to prevent the fading of chromophores. Best results can be achieved by imaging within the first week after staining. Some samples can be imaged even months after staining.

When imaging with the microscope first focus on the null sample using the DAPI channel, then increase or reduce laser straight and gains or light intensity and exposure time to the point that null sample nonspecific signals become barely visible. Thus cutting off the nonspecific signals. You can now proceed to image the rest of your samples, do not change imaging settings after setting up null. Do not change settings between the samples to ensure equal imaging conditions.

General comments:

1. Read antibody datasheets they are a good source of information on the best dilutions if available check the publications that using the antibodies for imaging it will help to know in advance how your target looks like in the cell. 2. Use ice to protect antibody vials during manipulations. The antibody is expensive. 3. Protect secondary antibody and samples that are applied secondary antibody from light and minimize the light exposure. 4. Many antibodies contain sodium azide, which is toxic and absorbs from skin – use gloves.

5. Antigen retrieval is usually not required when working with cryosection. But a must when working with paraffin sections. However, if you failed to achieve the specific signals you can use ph 9 hot (95c) citrate buffer incubation to improve the antigen reactivity. Heat up the citrate buffer in the microwave until boiling. Leave the samples in the hot citrate buffer for 10-15 minutes. Do not heat up citrate buffer with samples inside the boiling bubbles that may strip your specimen.

6. If you have issues with the adhesion of your sections coat the sample glass with poly-d lysine for 1-2 hours at room temperature or 4C overnight.

7. Don't overfix your specimens the 2% PFA overnight at 4C is usually sufficient to fix the brain. It is highly recommended to refuse the mice with saline and then with PFA to ensure proper fixation of the central regions of the brain and eliminate non specific signals from vasculature.

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