I am working on Immunohistochemistry on mouse brain and have sectioned 40 microns thick sections as per the protocol. I will be using 12-well plates. How many sections are advisable to be kept per well so that the wells are not over-crowded?
3ml is the volume of each well for the 12-well plates.
When performing immunohistochemistry on mouse brain sections in 12-well plates, it is important to ensure that the sections are not overcrowded to maintain proper antibody penetration and facilitate effective staining. The number of sections you can keep per well without overcrowding will depend on the size of your sections and the well size. Here's a general guideline you can follow:
For example, let's say the area of each well is 9 cm² (based on a volume of 3 ml and a well depth of 1 cm), and the average area of your sections is 0.25 cm². In this case, you could theoretically place a maximum of 36 sections (9 cm² / 0.25 cm²) per well without overcrowding.
However, it's important to consider practical factors such as ease of manipulation, antibody penetration, and staining quality. Therefore, it is advisable to leave some space between sections to allow for proper antibody penetration and staining efficiency. Depending on your specific experiment and staining requirements, you may want to reduce the number of sections per well to ensure optimal staining quality.
My own expiernce with free floatating immunohistohemistry:
Mouse tissue: 24 well plate; solution volumen 500µl/well -- max 3 sections/well
Rat tissue: -- max 1 Section/well
Mouse tissue: 12 well plate; solution volumen 1000µl/well -- max 6 sections/well
Rat tissue: -- max 3 sections/well
Mouse tissue: 6 well plate; soluton volumen 300µl/well -- max 12 sections/well
Rat tissue : -- 8-10 sections/well
The reaction in this well plates should carried out on a rocking table.
During the agitaton the sections are rinsed by the antibody solutions. This guarantees a homogenious staining result
Thank you very much Dr. Saif Wahid & Dr. Ute Neubacher for your guidance.
I have used 2-3 coronal sections per well in 24well plate. I recommend you to first place the sections and check if they are not overlapping. Also, make sure to put enough reagents to cover the sections. Good luck!
Hi Priyadarshini Dutta ,
This is a really interesting question.
In my experience, probably the factor affecting the most the quality of staining when using plates for Ab incubations is the type of shacking. Assuming everything else works properly.
Let's say 6 mouse-brain slices in 1mL solution (P12 plate):
In a rocker, the slices will substantially overlap during most of the incubation period (because they will end up sinking). While in a microplate orbital-shaker, you can maintain the slices actually floating with mild agitation during all the incubation period, minimizing a lot the time of actual overlapping of the slices. This allows a more equal distribution of the antibody over the tissue, even with higher numbers of slices per well.
I usually use the rule of thumb of 1 section/100 uL antibody solution (in 12-wells plates), ensuring optimal shaking conditions.
However, I usually transfer the slices to a P6 plate for all the washes that only have PBS-Triton or just PBS to ensure a proper wash out of the non-specifically bound antibodies (by increasing, a lot, the volume of the washing solution).
Hope this helps,
Cheers,
J
Thank you very much for the guidance! Dr. Poornima D.E Weerasinghe-Mudiyanselage & Dr. J. Ramirez-Franco
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