The overall goal of this procedure is to get the cleared, intact tissue and visualize its three-dimensional molecular information using immunolabeling methods. This method can help answer key questions in the neuroscience and developmental biology fields such as neuronal connections, molecular categorization and structural change during development. The main advantage of this technique is tissue clearly enables sub-cellular level analysis of big specimens without sectioning.
To begin this procedure, incubate the fixed organs in A4P0 Hydrogel monomer solution at four degrees Celsius for 12 to 24 hours with gentle shaking. After that, add five milliliters of Hydrogel monomer solution to a 10 milliliter round bottom tube. Then, transfer the mouse brain sample to it and wrap the top of the tube with Parafilm.
Next, bubble nitrogen through the liquid with the Hydrogel infused brain sample for one minute. Quickly and tightly close the sample containing tube. Transfer the tube to a water bath for two hours.
After that, check the viscosity of the Hydrogel and then wash the polymerized sample briefly with 0.1 X PBS to remove excess Hydrogel. In this step, transfer the polymerized sample to a tissue container. And place the tissue container in the ETC chamber.
Next, fill the ETC chamber with ETC buffer using a peristaltic pump. Set the ETC conditions and turn it on. One to two millimeter thick brain slices are cleared with within two hours.
And a whole brain is sufficiently cleared within five to six hours. Subsequently, transfer the sample to a 50 milliliter conical tube with 45 milliliters of 0.1 X PBS. Wash the sample several times with 0.1 X PBS until no bubbles are seen when the tube is briefly shaken to confirm complete removal of SDS.
To perform c-PRESTO, transfer a small sample into a 1.5 milliliter tube. Add 500 microliters of antibody dilution solution with a dilution factor of one to 500 for collagen type four antibody and centrifuge the tube at 600 times g for two hours. After that, wash the stained sample with 0.1 X PBS by centrifugation at 600 times g for 30 minutes.
Then, add 500 microliters of antibody dilution solution with a dilution factor of one to 500 for Cy3 conjugated Anti Rabbit secondary antibody and centrifuge it 600 times g for two hours. Subsequently, wash the stained sample with 0.1 X PBS by centrifugation at 600 times g for 30 minutes. To perform s-PRESTO for larger tissue, transfer the sample into a three-way valve connected syringe in the closed valve position.
Add five to seven milliliters of antibody dilution solution with a dilution factor of one to 500 for collagen type four antibody. Subsequently, add the antibody solution into the sample by opening the valve. Next, in the open valve position, set the syringe piston to the 17 milliliter position to provide sufficient room for the infusion withdrawal movement.
Then, close the three-way valve after the syringe setting is finished. Place the syringe containing the sample on a syringe pump. Set the syringe pump conditions to an infusion withdrawal volume of ten milliliters per minute and a four minute pause time on continuous cycle mode.
Afterward, run the syringe pump for three to 24 hours at room temperature. Afterward, open the three-way valve and replace the solution with 0.1 X PBS. Wash the stained sample twice with 0.1 X PBS for one hour each time, using the syringe pump.
Then, replace PBS with the antibody dilution solution containing Cy3 conjugated Anti Rabbit secondary antibody and run the syringe pump for three to 24 hours. Following that, open the three-way valve and replace the solution with 0.1 X PBS. Prior to imaging, incubate the stained sample in an appropriate amount of cubic mount solution for one hour at room temperature with gentle shaking.
After an hour, replace the solution with fresh cubic mount solution and incubate for an additional hour. Since antibodies can not penetrate into dense tissues by diffusion, PRESTO immunolabeling methods are designed to actively infuse macromolecules and to deliver reagents into dense tissues. The application of centrifugal forces using a standard table top centrifuge markedly facilitated the delivery of antibodies.
Applying convection flow with a syringe pump also improved antibody penetration. For s-PRESTO, a syringe pump was used to deliver the antibodies. The mouse organs, such as kidneys and liver, were labelled with collagen type 4.
Compared to the conventional methods, three hours of c or s-PRESTO markedly enhances the labeling depth. In the kidney and liver, the depth of the z-axis reaches 300 to 350 micrometers or deeper in PRESTO samples, while control samples are labelled at a depth of only 40 to 50 micrometers. Once mastered, these techniques can be done in a day for tissue clearly if it is performed properly.
While attempting this procedure, it's important to remember to keep the time of tissue fixation Hydrogel monomer emulsion and electrophoretic tissue clearly. Following the procedure, other methods like immunolabeling can be performed in order to answer additional questions like analysis of cellular dynamics and identification of a neuronal socket across multiple organs. After its development, this technique paved the way for researchers in the field of medical science to explore disease diagnosis.
After watching this video, you should have a good understanding of how to do Hydrogel embedding and tissue polymerization. Don't forget that working with Hydrogel monomer solution can be extremely hazardous and precaution should always be taken while performing this procedure.
