Bloxall blocking solution

Deparaffinization sections were washed thrice for 5 min each with phosphate buffered saline (PBS) (pH 7.4). For antigen activation, Proteinase K (Worthington Biochemical Co., Lakewood, NJ, USA)/distilled water (0.2 mg/mL) was added dropwise to the sections, which were then incubated for 15 min. After rewashing with PBS, endogenous peroxidase activity was blocked by BLOXALL blocking solution (Vector Laboratories, Newark, CA, USA) for 10 min. Blocking used 0.05% normal goat serum/PBS solution for 1 h. After rewashing again with PBS, the following primary alternatives were reacted at 4 °C overnight: antitumor necrosis factor-alpha (TNF-α), rabbit polyclonal antibody (1:200 dilution, AF7014; Affinity Biosciences, Solon, OH, USA), and anti-interleukin-6 (IL-6) rabbit polyclonal antibody (1:100 dilution, ab6672; Abcam, Cambridge, MA, USA). Subsequently, the streptavidin-biotin-peroxidase complex technique was performed using an ABC kit (Vector Laboratories). After rewashing with PBS, the sections were colored brown using a 3,3-diaminobenzidine solution (NICHIREI Biosciences, Tokyo, Japan). Nuclei were counterstained with Mayer’s hematoxylin. Immunohistochemically (IHC) stained images were calculated as the ratio of positive cells per unit area using the Fiji image analysis software [18 (link)].

Serial four-micrometer tissue sections were cut from the TMA blocks containing cores of fibroid and tumor margins and applied to special coated slides. Rabbit polyclonal antibody to Vitamin D3 Receptor (Biorbyt orb214726) was used to demonstrate the antigens present in the tissue material (Figure 3). Sections treated with a matched concentration of non-immune IgG were used as a negative control. Spleens were used as a positive control for immunohistochemical staining and localization.
Slides were incubated in a water bath at 96 °C in citrate buffer at pH 6.0 for 50 min. Endogenous peroxidase activity was blocked with 3% H₂O₂.
Tissue material was incubated with the ImmPRESS^® Horse Anti-Rabbit IgG PLUS Polymer Kit -ImmPRESS Horse Anti-Rabbit IgG Polymer Reagent and BLOXALL^® Blocking Solution (Vector Laboratories Inc., Burlingame, 94010 CA, USA, Catalog no.: MP-7801) for 30 min. The antigen was localized using Chromogen DAB-3.3 in all preparations. The antibody stained the cell nuclei in brown. The membrane and cytoplasm of each cell were unstained (Figure 3).

Unstained slides from liver, spleen, uterus, and mesenteric lymph nodes were adhered to positively charged glass slides for immunohistochemistry. Slides were deparaffinized and rehydrated through a series of xylene and ethanol steps before antigen retrieval was performed using 1:10 EMS Solution A (Electron Microscopy Services) in a 2100 Antigen Retriever (Aptum Biologics Ltd.), according to the manufacturer’s instruction. Endogenous peroxidase activity was blocked by 10 m incubation with Bloxall Blocking Solution (Vector Laboratories) followed by 20 m blocking with normal goat serum (Vector). After each step slides were washed with PBS plus 0.5% tween for 5 minutes. Primary incubation was overnight at 4°C with Brucella polyclonal rabbit antibody (Bioss) at 1:400. Negative control tissues were incubated with rabbit nonimmune serum diluted in PBS. A Vectastain ABC and Betazoid DAB chromagen kits (Biocare Medical) were used following primary incubation according to the manufacturer’s instructions. The slides were counterstained with Meyer’s hematoxylin III.

For immunohistochemical analysis, 3 μm sections of F2 fish of 3 wpf, 5 fish per genotype (wt, npc1^+/−, npc1^−/−) of npc1^Δ56 and npc1^Δ7 lines were dewaxed and rehydrated at room temperature in a humid chamber. Slides were washed with 10 mM phosphate-buffered saline and 0.5% Tween 20 (pH 7.4) in three successive 5 min immersions. Endogenous peroxidase was quenched with Bloxall Blocking Solution (Vector Laboratories, Inc.; Burlingame, CA, United States) and the sections were incubated with a primary rabbit monoclonal recombinant anti-Niemann Pick C1 antibody [EPR5209] (ab134113 Abcam; Cambridge, United Kingdom) generated against the C-terminal region of the protein. Immunohistochemical staining was carried out with ImmPRESS HRP Horse Anti-Rabbit IgG Polymer Kit (Vector Laboratories, Inc.), together with the Vector VIP Substrate Kit (Vector Laboratories, Inc.) as chromogen. Negative controls were included in the experiment by using the antibody dilution solution without primary antibody. Sections were counterstained with hematoxylin.

Samples were collected and fixed with 4% paraformaldehyde in PBS. After fixation, tissues were embedded in paraffin, before cutting into 4 µm sections and staining with hematoxylin and eosin (H&E) or performing immunohistochemistry (IHC) with rabbit anti-UCP1 antibody (Abcam, ab10983)^{6 (link)}. For IHC, rehydrated sections were microwaved in 10 mM citric acid buffer (pH 6.0) for antigen retrieval, and endogenous peroxidases were quenched with BLOXALL Blocking solution (Vector Laboratories, Burlingame, California). Sections were blocked with Avidin D, biotin and protein blocking agent in sequential order followed by application of the anti-UCP1 antibody (1:500). A biotinylated anti-rabbit antibody was used as a secondary antibody. Horseradish peroxidase-conjugated ABC reagent was applied, and then DAB reagent was used to develop the signal before counterstaining in hematoxylin and dehydrating the sections in preparation for mounting. Stained sections were acquired using a Leica DME microscope and Leica ICC50HD camera (Leica, Wetzlar, Germany) and analyzed using Leica LAS EZ software. Adipocyte size quantitation was performed using the Adiposoft plugin of ImageJ (NIH, Maryland, USA).