Bloxall sp 6000

IF staining was performed for p-ERK1/2 and p-PI3K on tumor tissue sections. After heat-induced epitope retrieval in a pH 6 buffer, sections were washed twice with buffer (1× TBS/0.1% Tween-20). Endogenous peroxides were blocked with BLOXALL (SP-6000, Vector Labs, Burlingame, CA). Tumor tissues were washed 2× with buffer solution, then incubated with blocking solution (5% Normal Goat Serum/wash buffer) for 1 h at room temperature and washed again. Primary antibodies (anti-pERK1/2 #14-9109-82, Thermo-Fisher, and anti-pPI3K #MA1-74183 Thermo-Fisher at dilutions of 1:250) were applied in blocking solution, and sections incubated overnight at 4 °C in a humidified chamber, followed by multiple washes and addition of Alexa-Fluor 555 conjugated anti-mouse secondary antibody (Cell Signaling, #4412) at a dilution of 1:1000 in blocking solution. Tissue sections were incubated at room temperature for 30 min and washed in triplicate for 5 min. Nuclear counterstaining was performed with ProLong Gold Antifade Mountant with DAPI (Invitrogen, #P36931).

Immunohistochemistry of mice muscle sections (Wt and G93A*SOD1) for SOD1 was used to observe the expression pattern of mutant-SOD1 in muscle sections, as described previously [55 (link)]. Briefly, d thin serial sections of 5 μm from paraffin-embedded tissues were subjected to subsequent deparaffinization, hydration, antigen retrieval (H-3300, Vector Laboratories, Burlingame, CA, USA), blocking of endogenous peroxidases (0.3% v/v hydrogen peroxide; Bloxall, SP-6000, Vector Laboratories, Burlingame, CA, USA), blocking with 5% serum (Vector Laboratories, Burlingame, CA, USA), and primary antibody incubation in blocking serum overnight. The sections were incubated with secondary antibody (Vector Laboratories, Burlingame, CA, USA) amplified antigen-antibody interaction using VECTASTAIN Elite ABC Peroxidase kit (PK-6100, Vector Laboratories, Burlingame, CA, USA) and visualized using DAB-chromogen System (SK-4105, Vector Laboratories, Burlingame, CA, USA). Subsequently, the sections were counterstained with hematoxylin, mounted using Cytoseal XYL mounting medium (Thermo Scientific), and imaged using Olympus BX40 microscope (Olympus Life Science, Waltham, MA, USA). The primary antibody used in this experiment is SOD1 (1:100; 37385; Cell Signaling).