Recent studies have highlighted the critical role of angiogenesis and sympathetic innovation in Adipose tissue remodeling during the development of obesity. Here we demonstrate a modified immunofluorescence method that efficiently stands blood vessels and nerve fibres in Adipose tissue. Our approach is straight forward and efficient with no compromise on standing efficiency and the quality.
We acquire the images using confocal microscopy. The high resolution of the images allows us to reconstruct the blood vessel networks and accurately analyse its characteristics. Begin this procedure with anesthesia and perfusion of six week C57 Black-6J male mice as detailed in the text protocol.
Dissect white and brown adipose issues from the mice. Use scissors to break the tissue samples into small pieces of chunk. Transfer the small chunks with the sterilized tweezers into the tissue embedding cassettes with proper labeling of the tissue samples on the cassettes.
Immerse the embedded cassettes containing tissue samples into the fixation solution at four degrees celsius for 24 to 48 hours. Transfer the small chunks was sterilized tweezers into a Petri Dish. Wash the samples 3 times with brush 1x PBS buffer at 0.5 milliliters per wash.
Now cut the fixed tissue samples into approximately two cubic millimeter cubes with the sterilized scissors. For permeabalisation, transfer the samples into a 1.5 milliliter tube containing one milliliter of 1%Triton PBS buffer and gently rotate the tubes at room temperature for 1 hour at 18 RPM. After an hour carefully remove the 1%Triton PBS buffer by aspiration wash the samples three times by adding the 1x PBS directly into the same tubes.
During each wash, invert the tubes several times. For blocking, add 0.5 milliliter of blocking buffer to the samples and incubator at room temperature for two hours with gentle rotation. To prepare 0.4 milliliters of primary antibody solution, dilute two microliters of anti Endomusin and two microliters of anti-tyrosine hydroxy lase antibodies until 396 microliters of blocking buffer.
Vortex and spin down to recover the volume. Now, carefully remove the blocking buffer from the tissue chunks. Add 100 microliters of the prepared primary antibodies solution into tubes and incubate at four degrees celcius overnight.
The next day, carefully collect the primary antibody solution for reuse if desired. Wash the samples three times with 1x PBST at 500 micro litres per wash for 30 minutes with gentle rotation at 18 RPM. For the preparation of secondary antibodies solution, dilute two microlitres of flouro-flouro conjugated anti-god IGG in two microlitres of flouro-flouro conjugated anti-rabid IGG into 396 microliters of blocking buffer.
Vortex and spin briefly to collect all the liquid. Now, remove the last wash buffer from the samples. Add 100 microliters of secondary antibody solution into tubes.
Incubate the samples at room temperature for two hours with gentle rotation at 18 RPM. After carefully removing the secondary antibody solution, wash the samples three times with 1x PBST at 500 microlitres per wash for 30 minutes each with gentle rotation at 18 RPM. For optical clearance, immerse the samples in one milliliter of 90%Glycerol and keep the samples at four degrees celcius in darkness until they become transparent.
Add here a silicon isolator to the slide to create a well for volume imaging. Keep the height of the silicone to or slightly less than that of the sample. Carefully transfer the samples into the well and fill it with the mounting medium.
Lay a coverslip on the surface and seal the quarters of the coverslip with high viscosity medium. Let the mounting medium cure for 24 hours at room temperature and darkness. After curing is complete, fully seal the edges of the coverslip for optimum sample longevity.
Acquire Z stack images with the 20 times objective of a confocal microscope and its corresponding software. Start the system. Click on the configuration tab and activate both the Argon laser and the He-Ne 633 laser.
Click on the acquire tab. In the visible laser lines panel, move the corresponding intensity sliders up to choose the laser lines up 488 nanometers and 633 nanometers. Initially the intensities can be set to 20 to 30%Now select the 488, 561, 633 triple dichroic mirror.
Activate the photo multipliers by clicking on the active check boxes. Choose photomultiplier one for the emission exerted by the 488 nanometer laser and photomultiplier 34 for that of the 633 nanometer laser. Set the wavelength range to between 500 and 550 nanometers for photo multiplier one.
In 650 to 750 nanometers, for photo multiplier three. Select the pseudo color to be used for image display by double clicking the colored rectangle beside each photo multiplier and choosing a color from the pop-up menu. Now click on live to check the image at the samples.
Use the Z position nob to select a plane of focus within the XY region of interest. Adjust the brightness of the images by turning the laser intensity, smart gain, and offset. For each fluorescence channel, perform this adjustment under the quick lookup table display mode.
Click on the quick look up table button to enter the quick lookup table mode in which saturated pixels are displayed in blue to aid the setting of appropriate brightness level. Click twice on the quick look up table button to go back to the pseudo color display mode. While scanning, turn the Z position nob counter clockwise to move the plane of focus to one end of the volume of interest.
Then click on the end arrowhead to set the scanning and position. Turn the Z position nob of clockwise to move the plane of focus through the specimen to the other end of the volume of interest. Then click on the begin arrowhead to set the scanning begin position.
Adjust the Z step size to three microns. Change the image quality by selecting a format of 1024 by 1024, A speed of 100 Hz, and a line average of two. Select start to initiate the Z stack image acquisition For maximum projection of the acquired image stack, Click on the process tab and then tool;select 3D projection and enter maximum in the method list without changes to X, Y and Z.Set threshold to zero, click on apply.
The maximum intensity of the Z volume will be stacked into a 2D image which is displayed. To analyse the 2D images via open source software, open the stacked images in the software. Adjust vessel diameter and intensity until all vessel structures can be properly selected.
Select run analysis and export the data following guidance of the software. To analyse the 3D images via the licence software, open the raw data of images with the software. Adjust the intensity threshold and other parameters until the vessel structures in each layer of the Z volume are properly segmented.
Skeletonize the resulting binarised image stack to obtain a special graph. Analyse the selected segment characteristics and export the data following the guidance of the software. The distal region epididymal white adipose tissue, the medial region of the dorsal lumbar subcutaneous white adipose tissue and the medial region of the intra-scapular brown adipose tissue were collected.
After a whole mount staining, the tissue trunks were mounted and imaged with the confocal microscope. More blood vessels were positively stained. With the anti-Endomusin antibody in the glycerol incubated subcutaneous white adipose tissue, suggesting that the clearing step is critical for complete staining of the blood vessels.
To determine whether blood vessels and nerve fibers exhibit different patterns among the depos with this method, Communal fluorescence staining was performed in adipose tissue with antibodies with anti-Endomusin to show blood vessels and with anti-tyrosine hydroxy lase to show nerve fibres. Interestingly, results show that they were significantly more blood vessels than nerve fibres in brown adipose tissue as compared to white adipose tissue. Among the white adipose tissue, the subcutaneous white adipose tissue exhibited higher blood vessel density than the epididymal white adipose tissue.
Of note, while the nerve fibres expanded in parallel with the blood vessels, they did not show significant co-localization. The vessel area, number of junctions, and tube length were then qualitatively measured with the 2D method in these three types of adipose tissue and found similar results. This method efficiently cos-dens blood vessels and nerve fibers and adipose tissue.
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