Bsa blocking buffer

We homogenized frozen brain tissues (175 ± 20 mg) in 5% glucose in distilled water in grinding tubes (Bio-Rad, Hercules, CA, USA) adjusted to 10% (wt/vol) by using a TeSeE Precess 48TM homogenizer (Bio-Rad), according to the manufacturer’s instructions. We determined presence of PrP^res in transgenic mouse brains by Western blot, using the reagents of the ELISA commercial test TeSeE (Bio-Rad). Based on a previously described protocol (31 (link)), we treated 10–100 μL of 10% wt/vol brain homogenates with proteinase K; the resulting samples were loaded in 12% Bis-Tris Gel (Criterion XT; Bio-Rad). We transferred proteins electrophoretically onto PVDF membranes (Millipore, Billerica, MA, USA), which were blocked overnight with 2% BSA blocking buffer (Sigma-Aldrich, St. Louis, MO, USA). For immunoblotting, we incubated with Sha 31 (44 (link)) monoclonal antibody (mAb) at a concentration of 1 µg/mL to identify the 145-WEDRYYRE-152 epitope of the human PrP^C sequence. To detect immunocomplexes, we incubated the membranes for 1 h with horseradish peroxidase conjugated anti-mouse IgG (GE Healthcare Amersham Biosciences, Little Chalfont, UK). Immunoblots were developed with enhanced chemiluminiscence ECL Select (GE Healthcare Amersham Biosciences). Images were captured using the ChemiDoc WRS+ System (Bio-Rad) and processed using Image Lab 5.2.1 software (Bio-Rad).

For whole mount staining, E15.5 and E18.5 embryos were harvested, skinned, eviscerated, and fixed in 95% ethanol overnight followed by 95% acetone overnight. Specimens were stained with Alcian Blue for cartilage (0.03% in acetic acid/95% ethanol—1:5 ratio), cleared in 1% KOH, and subsequently stained in Alizarin Red for bone (0.01% in KOH).
For immunohistochemistry staining, tissue was harvested, fixed in formalin, decalcified in 12% ethylenediaminetetraacetic acid (EDTA) (pH 7.4), and paraffin‐embedded. Histological sections were cut 4‐μm thick and paraffin was removed in xylene, followed by rehydration with a graded ethanol series (100%, 95%, 90%, and 70%) for 3 min each. Specimens were treated with proteinase K for 15 min for antigen retrieval and incubated with 3% H₂O₂ for 15 min to quench endogenous peroxidase activity. Specimens were then blocked for 30 min using 1% BSA blocking buffer (Sigma‐Aldrich). Primary antibodies against mouse Metrnl and isotype controls including rat immunoglobulin G (all antibodies where purchased from R&D Systems) were applied as per manufacturer's guidelines and incubated at 37°C for 2 h. This process was followed by incubation with a corresponding anti‐rat secondary antibody for 30 min at 37°C. Specimens were developed using DAB Substrate Kit (Vector Laboratories) and counterstained using Mayer's hematoxylin (Sigma‐Aldrich).