Cas block histochemical reagent

Paraffin-embedded heart sections were deparaffinized by introducing the slides in xylene followed by a wash with 100% ethanol. Both steps were carried out for five minutes. Antigen retrieval was done using a standard citrate buffer protocol. [38 (link)] Heart sections were blocked by CAS-block Histochemical Reagent (Thermo Fischer Scientific, USA) for 2 hours at room temperature followed by three PBS washing with 5 minutes interval between each wash. Primary antibodies for von Willebrand Factor (vWF) (catalog # A0082, Dako, Agilent Pathology Solutions, USA) and smooth muscle actin (SMA) (catalog # A2547 SIGMA, Sigma-Aldrich, USA) were used at a working dilution of 1:200 with CAS-Block. Both antibodies were individually incubated overnight at 4°C followed by three PBS washing with 5 minutes interval within each wash. The corresponding secondary antibodies were used at a working dilution of 1:200 with CAS-Block and individually incubated for 2 hours at room temperature followed by three PBS washing with 5 minutes interval within each wash. The nuclei were stained using DAPI (Sigma-Aldrich, USA). Images were analyzed using fluorescent microscopy. The results were compared by student’s t-test and a p value ≤ 0.05 was considered as significant. Results were represented as mean ± standard deviation.

Paraffin-embedded heart sections were deparaffinized by soaking the tissue sections alternatively in xylene and ethanol solutions (100%) for 5 mins each. Antigen retrieval was then performed using a citrate buffer solution and samples were then blocked using a CAS-block Histochemical Reagent (Thermo Fischer Scientific, USA) at room temperature for 2 h. This process was followed by three consecutive washes with PBS (5 min each). Samples were then incubated overnight at 4 °C with primary antibodies for von Willebrand Factor (vWF) (catalog # A0082, Dako, Agilent Pathology Solutions, USA) and smooth muscle actin (SMA) (catalog # A2547 SIGMA, Sigma-Aldrich, USA).^{52 ,53 (link)} Primary antibodies were used at a working dilution of 1:200 with CAS-Block. After washing samples with PBS three times (5 min each), they were incubated with the corresponding secondary antibodies (working dilution of 1:200 with CAS-Block) for 2 h at room temperature. DAPI (Sigma-Aldrich, USA) staining was performed to stain nuclei and images were obtained using fluorescent microscopy.¹⁸

Slides with tissue sections were washed three times in PBS for 10
minutes, blocked 1 hour in CAS-Block Histochemical Reagent (00-8120, Thermo
Fisher Scientific), incubated with primary antibodies overnight at
4°C, washed three times in PBS for 10 minutes, and then incubated
with secondary antibodies at for 1 hour at room temperature. Slides were
then washed twice in PBS for 10 minutes and then for 10 minutes with a PBS
containing DAPI (D9542, Sigma-Aldrich). Lastly, slides were mounted using
Southern Biotech Fluoromount-G (010001, VWR) and sealed. Antibodies
used for IF: Rabbit anti-Tubb3 (1:1000, AB18207, Abcam), Chicken
anti-mCherry (1:1000, AB356481, EMD Millipore), Goat anti-Ass1 (1:1000,
ab77590, Abcam), Rabbit anti-Celsr3 (1:500, SAB4500707-100UG, Sigma), Rabbit
anti-Uchl1 (1:800, 13179T, Cell Signaling), Rabbit anti-ASL (1:200, ab97370,
Abcam), Rabbit anti-GRP (1:1000, ab22623, Abcam), Rabbit anti-Prph (1:2000,
ab4666, Abcam), Goat anti-Phox2b (5 μg/mL, AF4940, Novus Biologicals)
and Alexa Fluor 488-, 594-, and 647-conjugated secondary antibodies (Life
Technologies) were used.