Image pro plus image analysis system

The BLA was prepared for western blotting analysis using Syn‐PER Synaptic Protein Extraction Reagent (Thermo Fisher, USA), resulting in both a functional synaptosome fraction and a cytosolic fraction.
²¹ (link) The protein concentration was determined by using BCA. SDS–PAGE gels were used to separate the same amounts of proteins before transfer to PVDF membranes. The PVDF membrane was blocked with 5% skim milk or 3% BSA (bovine serum albumin) at room temperature for 2 h, and then incubated overnight at 4°C with anti‐SK2 (1:500; Alomone labs, Israel), anti‐PKA (1:1000 Abcam ab38949), anti‐pPKA (phospho S96, 1:1000 Abcam ab32390), anti‐MPP2 (1:1000; Abcam, USA), anti‐GAPDH (1:1000), or anti‐β actin (1:3000) primary antibodies. After washing with TBST, the PVDF membrane was incubated with an HRP‐conjugated secondary antibody (1:1000) for 45 min at room temperature. Protein bands were illuminated using the BeyoECL Moon kit and captured using the Image ProPlus image analysis system (Media Cybernetics, Inc., Rockville, MD, United States).

Rats were sacrificed approximately 2 h after adult CRD. The lower lumbar and upper sacral segment (L4–S4) was extracted and lysed with Syn-PER^TM Synaptic Protein Extraction Reagent (1 mL/100 mg) containing PMSF. Cell lysis was centrifuged at 8.000 rpm for 10 min at 4°C. The supernatant (whole cell lysis) was collected and further centrifuged at 15,000 rpm for 20 min at 4°C. The supernatant (cytoplasmic fraction) were collected. RIPA lysis buffer (500 μL/200 mg) was added to the pellet (membrane fraction). The sample protein concentration was determined with BCA. Equal amounts of protein were separated by SDS-PAGE gels and transferred to the PVDF membrane. After blockade with 5% non-fat milk for 2 h at room temperature, the PVDF membrane was incubated with anti-SK2 (1:200) anti-GAPDH (1:1,000) or anti-Tubulin β (1:3,000) primary antibody at 4°C overnight. After TBST washing, the PVDF membrane was incubated with AP-conjugated secondary antibody (1:1,000) or HRP-conjugated secondary antibody (1:1,000) for 2 h at room temperature. Protein bands were illuminated using the BCIP/NBT alkaline phosphatase color development kit or BeyoECL Moon kit and captured by using Image ProPlus image analysis system (Media Cybernetics, Inc., Rockville, MD, United States).

Immunocytochemistry was conducted as described previously [6 (link)]. Briefly, keloid fragments (5 mm × 5 mm × 5 mm) were fixed in 4% paraformaldehyde at 4°C for 48 h, embedded in paraffin, sectioned at 4 μm thickness by Rotary Paraffin Microtome Slicer paraffin embedding machine (Media Cybernetics, USA), and stained with hematoxylin and eosin for routine examination. Subsequently, the keloid sections were incubated with antibodies against p-PI3K (1 : 100, mouse anti-human PI3K monoclonal antibody, Santa Cruz, CA, USA), PTEN (1 : 100, mouse anti-human PTEN (a2b1) monoclonal antibody, Santa Cruz, CA, USA), p-Akt (1 : 100, mouse anti-human Akt monoclonal antibody, Santa Cruz, CA, USA), or p-mTOR (1 : 100, mouse anti-human mTOR monoclonal antibody, Santa Cruz, CA, USA). Bound antibodies were visualized using 3,3′-diaminobenzidine (DAB) as a chromogen (ZSGB Bio Co., Ltd, Beijing, China), and the slides were counterstained with hematoxylin. The positive expression of p-PI3K, PTEN, p-Akt, and p-mTOR was evaluated in four randomly selected fields under a light microscope (Olympus Corp., Tokyo, Japan). The mean optical densities (MOD) of positive expression were quantified with the Image Pro-Plus image analysis system (Media Cybernetics, Inc., Rockville, MD, USA).

Immunohistochemical staining was performed using PV two-step immunohistochemical detection kit (Beijing Shan Jinqiao Biological Technology Co., Ltd., Beijing China), integrin αv, vinculin and connexin43 positive cells were detected using mouse anti-rat integrin αv monoclonal antibody (1:50), mouse anti-rat vinculin monoclonal antibody (1:300) and rabbit anti-connexin43 polyclonal antibody (1:200) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) respectively. Briefly, sections were incubated with 3% H₂O₂ at 37°C for 30 min, repaired by microwave, blocked with dripped 10% goat serum for 30 min, incubated with primary antibody at 4°C overnight, visualized with a PV two-step immunohistochemical detection kit, and re-stained with hematoxylin (Suzhou Jin Pure Chemical Co., Ltd.). The Image-Pro Plus image analysis system (Media Cybernetics, Inc., Rockville, MD, USA) was used to analyze the average optical density (OD).