This free floating technique can be used to perform for lessened immunohistochemistry on mouse brain tissue samples. The free floating technique provides several advantages. The acquisition of thicker tissue sections, the performance of large scale studies, using 30 to 40 slices per aliquot and the long-term storage of samples.
If you are trying this technique for the first time, we recommend you practice mounting sections that are not critical to your experiment to obtain experience with the method. At least one to two hours before sectioning, acclimatize the samples of interest in the cryostat at the appropriate tissue cutting temperature. When the tissues are ready.
use the cryostat to acquire 20 to 50 micron thick sections and place the sections into individual inserts in PBS filled wells of a six or 12 well plate. When all of the sections have been acquired, cover the samples with approximately six milliliters per wash for three five minute washes before transferring each well of sections into two milliliter micro centrifuge tubes, containing one to 1.5 milliliters of storage solution per tube. Then store the samples at minus 80 degrees Celsius until staining.
For tissue section staining, equilibrate the samples to room temperature for 10 to 20 minutes before decanting the sections into inserts in a new six well plate. When all of the storage solution has been drained, move the inserts into new wells, containing approximately six milliliters of TBS per well for three washes for five minutes with fresh TBS per wash on an orbital shaker at low speed at room temperature. After the last wash, add seven milliliters of freshly prepared blocking permeabilizing solution to each well for a 30 minute incubation on the shaker at room temperature.
At the end of the incubation, add one milliliter of the primary antibody solution of interest to one two milliliter micro centrifuge tube per insert and transfer all of the sections in each insert into the appropriate two milliliter tube. Then place the tubes on a rotating mixer. Add about seven revolutions per minute for an overnight incubation at four degrees Celsius.
The next morning, decant the sections from each tube into individual well inserts in a new six well plate and wash the sections two times for 30 seconds per wash and one time for 10 minutes in fresh TBS per wash. After the last wash, transfer the groups of sections into individual two milliliter micro centrifuge tubes, containing one milliliter of the appropriate secondary antibody solution per tube and incubate the samples for two hours at room temperature on an orbital shaker at low speed, protected from light. At the end of the incubation, decant the sections into inserts in a new six well plate and wash the samples two times for 30 seconds and one time for 15 minutes in fresh TBS per wash, protected from light.
After the last wash, decant the sections into a glass rectangular histological chamber, three quarters filled with TBS and submerge a glass slide in the TBS. Using a fine paintbrush and a magnifying lamp if desired, carefully coax the sections toward the slide and gently tap a section onto the slide, making sure there are no wrinkles or folds. When all of the sections have been captured onto a slide, allow the slides to dry for about 10 to 15 minutes at room temperature, protected from light, until the sections are opaque and apply an appropriate aqueous mounting medium to the sections.
Use tweezers to place a cover slip onto the medium and use a piece of filter paper to firmly press down on the cover slip to remove any excess medium. The sections can then be imaged, using an appropriate microscope or stored in a dark slide box set four degree Celsius. As observed in these representative images, fluorescent immunohistochemical analysis, using the free floating method can be performed on mouse brain tissue sections to examine glial fibrillar acidic protein expression.
This approach is also appropriate for revealing low expression proteins, such as GFP, in a low expressing transgenic mouse brain sample or in non fluorescent histochemical staining protocols, such as cresyl violet. Other peripheral tissues are also amenable to use of this technique with no modifications required as observed for these low level GFP expressing liver tissue samples. The most important thing to remember with the free floating technique is to be gentle with the sections, especially during transfers and when the samples are on the shakers or rotators.
In addition to a immunofluorescence staining, this method can be easily modified for other histochemical stains such as hematoxylin and eosin and cresyl violet or for chromogen immunohistochemistry.
