Diaminobenzidine solution

A549 cells transfected with si-NC and si-FAM188B and cells (1 × 10⁶/100 μL) were injected into the tail vein of BALB/c nude mice (n = 5 in each group). The mice were sacrificed after eight weeks, lungs were harvested, and the number of tumor nodules in the lungs was counted. Lungs were fixed in 4% paraformaldehyde and stained with hematoxylin-eosin staining solution (Sigma, St. Louis, MO, USA). The tumor tissues incubated with primary antibodies for 1 h were then treated with anti-rabbit biotinylated antibody (1:1000 dilutions; Vector Laboratories, San Francisco, CA, USA) for 1 h. Color reaction was developed by incubation with diaminobenzidine solution (Sigma) followed by counterstaining with hematoxylin. Stained tissues were reviewed by two experienced pathologists. This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Cancer Center Research Institute (NCCRI), and IACUC approval number is NCC-16-231. NCCRI is an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) accredited facility and abides by the Institute of Laboratory Animal Resources (ILAR) guide.

Tissue arrays were obtained from Superbiochips Laboratories (Seoul, Korea) as described previously.^{48 (link)} Each slide contained section of tumor and normal tissues obtained from cancer patient by biopsy or surgical resection. The tissues incubated with primary antibody for 1 h were then treated with anti-mouse biotinylated antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h. Color reaction was developed by incubation with diaminobenzidine solution (Sigma-Aldrich, St. Louis, MO, USA) followed by counter staining with hematoxylin. Parallel sections incubated with normal IgGs instead of primary antibodies were used as negative controls. The overall staining results were scored from 0 to 3 based on the intensity and positive rate of staining. Intensity of staining was categorized as 0, negative; 1, weak; 2, intermediate; 3, strong. Stained tissue arrays were reviewed by experienced pathologists.

After deparaffinization in xylene for 3 × 5 mins and rehydrated through descending concentrations of ethanol, the slides were placed into sodium citrate buffer to recover antigen in a microwave oven (15 min, 20 power). After incubating in 1% H₂O₂ for 10 min to inhibit endogenous peroxidases and blocking by using 5% bovine serum albumin (BSA) (Roche, 10,735,078,001, Switzerland) for 30 min at 37 °C, the sections were incubated at 4 °C overnight with polyclonal rabbit anti-kisspeptin primary antibody (Abbiotec, 251,265, USA) diluted 1:400, polyclonal rabbit anti-GPR54 antibody (Abbiotec, 254,512, USA) diluted 1:100 or polyclonal rabbit anti-FSHR antibody (Bioss, bs-20658R, China) diluted 1:400. Then, the sections were incubated for 30 min with biotinylated goat anti-rabbit secondary antibodies (Zhongshan Goldenbridge, PV-6001, China) at 37 °C. Finally, the reaction was visualized with diaminobenzidine solution (Sigma-Aldrich, USA). Primary antibody was replaced with phosphate buffer saline (PBS) as the negative control running in the same batch. All results were imaged on a light microscope (Olympus BX53, Japan). Semi-quantitative evaluation of the immunostaining intensity was carried out using Image-ProPlus 6.0 (IPP 6.0) system and mean optical density (MOD) was used to represent the levels of protein expression.

Except where indicated, staining was done on 40-μm free-floating sections of a one-in-six series through the entire hippocampus and cortex. Tissue was blocked in 10 % normal serum from the species in which the secondary antibody was raised, with the addition of 0.1 % Triton X-100. Primary antibodies were diluted in 3 % normal serum with 0.1 % Triton X-100, and tissue incubated overnight at 4 °C. Biotinylated secondary antibodies were diluted in 3 % normal serum with 0.1 % Triton X-100 and were incubated for 2 h at room temperature. Enzyme detection was done using avidin-biotin substrate (ABC kit, Vector Laboratories, Burlingame, CA, USA) followed by color development in diaminobenzidine solution (Sigma-Aldrich, St. Louis, MO, USA). Antibodies and dilutions were as follows: rat CD11b (Serotec, Raleigh, NC, USA; 1:500); mouse CD68 (Serotec; 1:200); rabbit YM1 (Stem cell technology, Vancouver, BC, Canada; 1:400); rat Marco (Serotec; 1:500); mouse NeuN (Millipore, Temecula, CA, USA; 1:1000). After incubation for 24–48 h with appropriate primary and biotinylated secondary antibodies, tissue was treated with Vectastain ABC reagent (Vector Labs) and visualized with DAB reaction (Sigma-Aldrich). Controls included omitting primary or secondary  antibodies.

The kidneys were fixed in formalin, embedded in paraffin and stained with PAS according to standard protocols. To analyse the expression of Ki-67, slides of fixed and paraffin-embedded mouse kidneys were de-paraffinized using Xylol and descending concentrations of ethanol. Antigen retrieval was carried out by warming kidney slides in citrate buffer (10 mM, pH6) for 10 min using a microwave. After blocking with 3% H₂O₂ and Avidin and Biotin (Vector Laboratories, Inc.) for 15 min each, slides were sequentially incubated with the Ki-67 antibody (rabbit Ki-67 ab16667, abcam, 1:500 dilution, over night at 4°C) and after washing with PBS with biotinylated anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA; 1 h at room temperature). Kidney slides were labelled with ABC kit (Vector Laboratories, Inc.), and development was carried out using diaminobenzidine solution (Sigma Aldrich). Slides were counterstained with hematoxylin (Sigma-Aldrich), dehydrated and afterwards mounted with Histomount (National Diagnostics). Stained slides were scanned using a Slidescanner (Leica) and analyzed using the ImageScope software (version 12.0.1.5030, Aperio).

Mice were sacrificed 90 min after FC training or test. Brains were removed and immediately frozen in liquid nitrogen vapor. 20-µm coronal sections were prepared on a cryostat (Leica) and fixed in 4% paraformaldehyde. For c-Fos immunostaining, primary rabbit polyclonal antibodies against the c-Fos protein (sc-52, Santa Cruz Biotechnology, Dallas, TX, USA, dilution 1:500) and horse secondary antibodies against rabbit conjugated with avidin-biotin complex (ImPRESS reagent kit anti-rabbit, Vector Laboratories) were used. Sections were stained in 0.06% diaminobenzidine solution (Sigma). Brain sections were taken at a distance of −1.7 mm from the bregma. Whole section images were acquired through the fluorescence microscope scanner (Olympus, VS110).