The paraffin-embedded LIHC samples and corresponding peritumoral samples were obtained from Xiangya Hospital, Central South University. The ethics was approved by Xiangya Hospital, Central South University (No. 202205113). Immunohistochemistry (IHC) was performed using a universal two-step IHC staining kit (PV-9000, ZSGB-BIO, Beijing, China) according to the instructions. The primary antibody used in this study was anti-ENPP1 (1 : 500, ab223268, Abcam). The IHC results were identified according to the staining percentage and staining intensity.
Ab223268
Manufactured by Abcam
Sourced in United Kingdom
Ab223268 is a lab equipment product from Abcam. It is a high-quality instrument used for scientific research and analysis. The core function of this product is to provide accurate and reliable data for its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Pv 9000, by ZSGB-BIO (1 mentions)
Anti rabbit, by Jackson ImmunoResearch (1 mentions)
Ab7671, by Abcam (1 mentions)
Alexa fluor 555 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Ab7291, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ab181052, by Abcam (1 mentions)
Ecl system, by Merck Group (1 mentions)
4 protocols using ab223268
1
Immunohistochemical Analysis of ENPP1 Expression
2
Immunohistochemical Evaluation of ENPP1
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All IHC for ENPP1 were carried out using standard protocols on formalin-fixed, paraffin-embedded TMA sections. Once sections were deparaffinized and rehydrated, antigen retrieval was carried out by incubating the sections in Tris EDTA buffer (CC1 standard) for 1 hour at 95°C. The resulting TMA sections were incubated for 1 hour (Ventana Discovery platform) in the ENPP1 primary antibody (ab223268, Abcam) according to the manufacturer's guidelines. Next, primary antibody-bound tissue sections were incubated with the appropriate secondary antibody (anti-Rabbit, 111–035–003, The Jackson Laboratory) for 1 hour followed by Ultramap HRP and Chromomap DAB detection. H-scores for each core were calculated by multiplying the ENPP1 intensity (0–3, where 0 denotes no staining, 1 denotes low staining, 2 denotes medium staining, and 3 denotes high staining) by the percentage of coverage of the core (0–100) for a total scale of 0–300. H-scores were determined by an experienced pathologist (A. Delaidelli). Where multiple cores existed for the same sample/patient, the average score was taken between them.
3
Multimarker Immunofluorescence Staining
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Cells were counted and seeded on chamber glass slides (Millipore) at a density of 10,000 cells per well. Two days later, the medium was removed, and following a brief wash with PBS, cells were fixed using 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes at room temperature. Cells were next permeabilized and blocked in a solution of 2% BSA containing 0.2% Triton X-100 for 30 minutes at room temperature. Following 3 washes with PBS, cells were incubated with primary antibodies against selected targets of interest (manufacturer's recommended concentration in 2% BSA) overnight at 4°C. The following primary antibodies were used: Anti-ATP1A1 (ATPase; ab7671, Abcam), anti-CDH11 (71–7600, Invitrogen), anti-ENPP1 (ab223268, Abcam), and anti-Tubulin (ab7291, Abcam). The next day, following 3 washes with PBS, cells were incubated with AlexaFluor secondary antibodies (Goat anti-Rabbit IgG Alexa Fluor 488 #A-11008, Goat anti-Mouse IgG Alexa Fluor 555 #A-21422, Thermo Fisher Scientific) raised against the primary antibody species of interest, for 1 hour at room temperature. Finally, cells were washed 3 times with PBS and a coverslip was applied atop the cells with a small amount of DAPI-counterstain mounting medium (Vector Laboratories) and sealed using nail polish.
4
Validating Key Cardiac Proteins
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Key differentially expressed proteins in the integrated metabolomic and proteomic analyses were validated using western blotting. In brief, cardiac valve samples were ground and lysed in RIPA lysis buffer containing protease inhibitor phenylmethylsulfonyl fluoride (PMSF), and protein concentrations were determined using a BCA Protein Assay kit. Proteins were separated on 10% polyacrylamide gels and transferred onto PVDF membranes. Non-specific antigen binding on the membranes was blocked using 5% skim milk in phosphate-buffered saline (PBS) for 1–2 h, and then washed three times using PBS-T buffer (1 × PBS + 1 mL of Tween-20). The membranes were incubated with anti-CaMKII delta (ab181052), anti-ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1)/PC-1 (ab223268), and anti-ATP Binding Cassette Subfamily A Member 8 (ABCA8) ABCA8 (ab230896) primary antibodies (all from Abcam, Cambridge, United Kingdom) at 4°C overnight, followed by HRP-conjugated secondary antibody. The membranes were washed with PBS-T buffer, and immunoreactive signals were detected using an ECL system (MilliporeSigma, Burlington, MA, United States). Gray values of protein bands were analyzed using the ImageJ software.
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