3 3 diaminobenzidine dab reagent

To assess colocalisation of 5-HT_2AR and POMC, brains taken from control rats, were first processed for detection of Htr2a mRNA by ISHH, as described above. Following this, the tissue was washed in PBS before commencement of the IHC protocol to label α-melanocyte-stimulating hormone (α-MSH) protein using procedures previously described (Alon et al., 2009 (link); Garfield et al., 2012 (link)). Briefly, sections were incubated in 0.3% H₂O₂ in PBS, then rinsed in PBS, blocked in 0.5% BSA/0.5% Triton X-100 in PBS and left in blocking buffer containing rabbit α-MSH antibody (1/10,000; Chemicon, Millipore, MA, USA) overnight. Tissue was then washed in PBS and a biotinylated donkey anti-rabbit secondary antibody (Vector Laboratories) was applied at 1:1000 in blocking buffer. Sections were then washed in PBS, incubated in VectaStain ABC reagent, and following this, chromogenic detection was conducted using 3,3′-diaminobenzidine (DAB) reagent (Vector Laboratories). Sections were mounted onto superfrost slides, dried, then dipped in photographic emulsion (Kodak) and stored at 4°C for 2 weeks before being developed using Kodak developer and fixer. Double-labelled cells were recorded if α-MSH-immunoreactive (IR) cell bodies were overlaid with a [³⁵S]Htr2a signal greater than 3× background.

Samples portioned for histological analysis were cryoembedded, sectioned at 14 μm, and fixed in 10% formalin. To visualize collagen and GAG distribution, samples were stained with picrosirius red and Safranin-O/Fast Green, respectively. Alizarin Red staining was used to assess mineralization. For Phase 2, Safranin-O/Fast Green images were used to quantify cell diameter using ImageJ software; specifically, 6 cells from each of 8 images were assessed for a total of 48 measurements per group. While tissue volumetric changes may occur during embedding, all samples were handled identically. As a result, calculated cell diameters apply only for cryoembedded, formalin-fixed samples. Immunohistochemistry was performed for collagen type II and X. Briefly, slides were fixed in acetone for 20 min at 4°C. Endogenous peroxidases were quenched with 3% hydrogen peroxide in methanol before blocking with 1% bovine serum albumin. The primary antibody (rabbit anti-collagen type II (Fitzgerald Industries International, Acton, MA, USA) and mouse anti-collagen type X (Abcam, Cambridge, UK)) was applied for 1 hr, followed by horseradish peroxidase-conjugated secondary antibody for 30 min (Vector Laboratories, Burlingame, CA, USA). Visualization of antibody localization was performed using 3,3′-diaminobenzidine (DAB) reagent (Vector Laboratories).