Bx 50f light microscope

Optimal cutting temperature (OCT) compound (Elkhart, IN, USA) was used for embedding adipose tissues, which snap frozen to − 80 °C. Then, it was cut into 5 μm thick sections with a cryostat (Leica, Bensheim, Germany), after which they were incubated with rabbit anti-UCP1 (absin, Shanghai, China) at a dilution of 1:500 overnight at 4 °C. The horseradish orseradish peroxidase-conjugated goat anti-rabbit IgG (Abclonal, Wuhan, China) was at a dilution of 1:2000 and incubated at 37 °C for 1 h. Streptavidin biotin complex (SABC) methods were employed to visualize. The Olympus BX-50F light microscope (Olympus Optical, Tokyo, Japan) was used to photograph the sections.

For IF assay, specimens were incubated at 4 °C overnight in 4% paraformaldehyde, embedded in paraffin, and sliced in 5 μm thick using a microtome (Leica, Bensheim, Germany). Slices were incubated with UCP1 antibody (anti-UCP1 rabbit polyclonal antibody; 1:500; purchased from Sangon Biotech, Shanghai, China; register number: D262447) overnight. Then, these slices were washed three times using phosphate buffer saline (PBS), incubated in the fluorescein isothiocyanate-conjugated secondary antibody (1:500; BOSTER, Wuhan, China; register number: BM2012) for 1 hour at 37 °C, and incubated in DAPI for 5 minutes. After being washed three times using PBS, all slices of IF were imaged using an Olympus BX-50F light microscope (Olympus Optical, Tokyo, Japan).

Perirenal fat were fixed with 4% paraformaldehyde and embedded in paraffin. For HE staining, sections were stained with hematoxylin (Solarbio, Beijing, China). Then, sections were photographed by the BX-50F light microscope (Olympus, Tokyo, Japan).

Five-μm-thick sections of paraffin embedded placenta, kidney, and uterus tissue blocks were serially cut, mounted on silanized slides, deparaffinized, and rehydrated in descending grades of ethanol. Selected levels of all tissues were then stained with hematoxylin and eosin (H&E) to evaluate general morphology, and selected levels of all kidneys were stained with periodic acid Schiff (PAS) reagent for the visualization of basement membranes of glomerular capillary loops and tubular epithelium. Histopathological examination of these tissue sections was performed on an Olympus BX50F light microscope (Olympus America Inc., Melville, NY, USA) by a pathologist (FQ). Kidney sections were evaluated for glomerular endotheliosis (e.g. ballooning of tips of capillary loops, capillary endothelial swelling, occlusion of glomerular capillaries) in at least 20 glomeruli in the inner cortex of one kidney in each animal.

Four-µm-thick sections were cut from paraffin embedded kidney blocks, mounted on silanized slides, deparaffinized and rehydrated. The general morphology was analyzed on selected tissue levels after staining with hematoxylin and eosin (H&E). Selected kidney sections were stained with periodic acid Schiff (PAS) reagent and with the Jones basement membrane reticulum stain (Dako Artisan Link Pro, Dako North America, Inc., Carpinteria, CA, USA) for the evaluation of the glomerular capillary loop basement membranes. Two pathologists (SJ and FQ) blinded to the clinical outcome evaluated 10 glomeruli from each kidney for glomerular endotheliosis (occlusion of glomerular capillaries, capillary tip ballooning, capillary tip swelling, and abnormalities of the capillary loop basement membranes) and changes in the mesangium. Glomerular damage was scored as follows: 0 = no glomerular changes in 10 glomeruli examined; 1+  = 1 to 5 of 10 glomeruli examined with either segmental or diffuse endotheliosis; 2+  = 6 or more glomeruli with segmental or diffuse endotheliosis. Images were taken with an Olympus BX50F light microscope (Olympus America Inc., Melville, NY, USA).

Samples of each fraction were smeared on glass slides (Muto Pure Chemicals, 5116-20F, Tokyo, Japan) using a wedge method and stained with Papanicolaou solution¹⁹ . The images were obtained either with a BX50F light microscope (Olympus, Tokyo, Japan) using 10 × and 20 × objective lenses (for the 0.3K, 2K, 10K, and 160K fractions) or with a Keyence BZ-X700 light microscope (Keyence, Osaka, Japan) using 20 × and 40 × objective lenses (for the OFs).