This method can help answer key questions in the adipose biology field, such as how intercellular interactions and adipose tissue physiology change upon different metabolic conditions such as fasting and obesity. The main advantage of this technique is that it optimally preserves the original adipose 3D structure and avoids lengthy processing steps that are usually required for conventional immunohistochemistry. Although this method can provide insight into the structure of the adipose tissue, it can also be applied to other organ systems, such as the nervous system, by whole-mount staining the brain.
Generally, individuals new to this method will struggle because of the difficulties in getting property tissue sites, finding the proper fixation period, and the optimal antibody concentrations. After dissection is complete, transfer the dish containing the tissue samples in 1%paraformaldehyde from the ice to room temperature for one hour. Then transfer the tissues to either a 12 or 24-well cell culture plate for quicker washing.
It's important to remember that the whole-mount staining procedure must begin immediately after dissection and that there are no pause points between each step. Wash the tissues with PBS-0.3T on a shaker tilted at 22 degrees with a speed between 20 and 25 tilts per minute at room temperature for five minutes. Repeat this wash two additional times.
After this, add approximately 0.5 to one milliliter of blocking buffer containing 5%animal serum. Incubate the plate on a shaker using the previous conditions for one hour. Aspirate the blocking solution, and add on milliliter of primary antibodies diluted in PBS-0.3T with 1%animal serum to each well.
Incubate the plate overnight on a shaker at four degrees Celsius with a tilt of 22 degrees and a speed of 20 to 25 tilts per minute. The next day, use PBS-0.3T to wash the tissues at room temperature for five minutes. Repeat this wash two additional times.
Then, add between 0.5 and one milliliter of appropriate and diluted secondary antibody solutions to each well. Wrap the plate in aluminum foil and incubate it on a shaker at room temperature for one hour. After this, wash the plate twice with PBS-0.3T at room temperature for five minutes per wash.
If visualization of liquid droplets is needed during the neutral lipid stain, wash twice with 1x PBS with each wash lasting five minutes. Add the neutral lipid stain diluted in PBS, and incubate at room temperature for 30 minutes. To begin imaging, use forceps to lay the tissue flat on a 24 by 60 millimeter glass cover slip.
If nuclei staining with DAPI is desired, add one to two drops of a mounting medium that contains DAPI to completely submerge the tissue and prevent it from drying. Then, place the slide on an inverted confocal laser microscope system. To obtain images of whole-mount stained tissue at multiple focal planes, perform Z stacks of 100 to 150 micrometers in depth with step size of four to six micrometers at the desired magnification.
In this study, whole-mount staining is used to preserve adipose tissue architecture. Unlike methods that involve multiple processing steps and sectioning, which can lead to disfigurement of the adipose tissue morphology, the whole-mount staining method can preserve the morphology of adipocytes, ensuring accuracy when interpreting results. Overfixation of adipose tissue leads to fixative-induced fluorescence, which can be indicated by overlapping identical regions, such as in the tyrosine hydroxylase and PECAM-1 signals seen here.
Properly fixed, whole-mount stain samples, however, show separate and distinct signals, indicating that these signals are not autofluorescence. Imaging whole-mount stained adipose tissue allows for the quantification of morphological changes under different experimental conditions. In particular, adipose tissue is highly vascularized, which is important in mediating metabolic homeostasis upon rapid changes in energy level.
For instance, C57 black 6J mice, that undergo 24 hours of fasting, display a significantly smaller adipocyte size, which indicates lipolysis, and a trend in elevated blood vessel density when compared to continuously fed mice. Clearing adipose tissues with iDISCO+enables adipose tissues to become optically transparent. Immunolabeling of adipose tissue that has gone through tissue clearing shows much denser neural arborization compared to that observed in whole-mount staining without tissue clearing at the same magnification.
While attempting this procedure, it's important to remember not to overfix your samples for more than a hour at room temperature in PFA, otherwise, this will increase background autofluorescence. However, this fixation period can be prolonged if samples are fixed at four degrees Celsius. Following this procedure, other methods like quantification using ImageJ, can be performed in order to answer additional questions, like how cellular architecture, such as adipocyte sites and blood vessel density, change in response to various physiological conditions.
