Four micrometers of formaldehyde‐fixed pleural tissue were dewaxed and rehydrated with an alcohol gradient and PBS. Antigen retrieval was performed with citrate (pH 6.0). Endogenous peroxidase was blocked with 3% H2O2 in water for 20 min, and nonspecific binding was blocked with diluted normal goat serum for 60 min, and was then incubated with the primary antibody (mouse anti‐human monoclonal antibody C1q (ab71089), rabbit anti‐human polyclonal antibody factor B (ab192577), rabbit anti‐human polyclonal antibody factor P (ab186834), mouse anti‐human monoclonal antibody MBL (ab23457), mouse anti‐human monoclonal antibody factor H (ab118820), mouse anti‐human monoclonal antibody C3a (ab37230), mouse anti‐human monoclonal antibody C5a (ab11877) and rabbit anti‐human polyclonal antibody SC5b‐9(ab55811), all antibodies were purchased from Abcam) for 18 h at 4°C. According to the manufacturer's instructions, labeling was identified using the SP goat IgG kit (PV‐6000, ZSGB‐Bio, China). The chromogenic reaction solution contained 3, 3′‐diaminobenzidine (DAB) (ZLI‐9018, ZSGB‐Bio, China), and counterstaining was performed with Mayer's hematoxylin (Solarbio). Slides were viewed under an imaging fluorescence microscope (Olympus BX51; Olympus).
Ab192577
Manufactured by Abcam
Sourced in United Kingdom
Ab192577 is a laboratory equipment product. It serves as a core function without additional interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Ab23461, by Abcam (2 mentions)
Ab83076, by Abcam (2 mentions)
Ab71089, by Abcam (1 mentions)
Ab11877, by Abcam (1 mentions)
Ab37230, by Abcam (1 mentions)
Ab186834, by Abcam (1 mentions)
Ab23457, by Abcam (1 mentions)
Ab118820, by Abcam (1 mentions)
Mayer s hematoxylin, by Solarbio (1 mentions)
Zli 9018, by ZSGB-BIO (1 mentions)
Streptavidin biotin kit, by Nichirei Biosciences (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs plus system, by Bio-Rad (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Ab11878, by Abcam (1 mentions)
Ab200999, by Abcam (1 mentions)
4 protocols using ab192577
1
Immunohistochemical Analysis of Complement System
2
Complement Pathway Analysis in Prostate Tissue
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Paraffin-embedded sections of rat and human prostate tissue were prepared and incubated with anti-C1q (ab171566 for rat, ab11861 for human), C3 (ab11887 for rat, ab129945 for human), MBL (ab23461 for rat and human), FB (ab192577 for rat and human), and C5b-9 (ab83076 for rat and human) primary antibodies (Abcam). Subsequently, the sections were incubated with appropriate biotinylated secondary antibodies. Staining was detected using a streptavidin-biotin kit (Nichirei, Tokyo, Japan.
3
Complement Protein Expression in Prostate Tissue
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Protein was extracted from frozen prostate tissues in RIPA Lysis and Extraction buffer (Thermo Fisher Scientific) and subsequently subjected to SDS-PAGE. Rabbit polyclonal anti-C1q (ab171566), C3 (ab11887), and FB (ab192577) antibodies and mouse monoclonal anti-MBL (ab23461) and C5b-9 (ab83076) antibodies were purchased from Abcam (Cambridge, UK) and used in the experiment as primary antibodies. The appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were used. Protein bands were visualized with ECLTM Advance Western Detection Reagent (GE Healthcare, Buckinghamshire, UK) and imaged with a ChemiDocTM XRS Plus System (BIO-RAD, Hercules, CA, USA). The membranes were re-probed with a rabbit anti-β-actin polyclonal antibody (CS 4967, Cell Signaling Laboratory, Danvers, MA, USA) and an appropriate secondary antibody to verify that protein loading was similar among the samples.
4
Immunohistochemical Analysis of Complement
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IHC method was employed to detect regional deposition of complement components, including C3 (ab200999, Abcam), C3a (neo-epitope, ab11873, Abcam), C5a (neo-epitope, ab11878, Abcam), fB (ab192577, Abcam), and CR1 (anti-CD35, ab25, Abcam). IHC results were analyzed by two experienced pathologists blinded to patient’s information, and were scored by a semi-quantitative method in which staining of more than 10% of tumor cells was considered positive. The staining intensity was scaled as 0 for negative, 1 for weak (10~40%), 2 for moderate (40~70%) and 3 for strong (> 70%). The average score of staining intensity was calculated with five independent high-power fields using IMAGE PLUS software (Version 6.0, Media Cybernetics, USA). Low and high C3 deposition were defined as ≤1 and > 1 point, respectively. Deparaffinized sections from harvested human tissues were pretreated with 10 mM sodium citrate buffer (pH 6.0, boiling temperature, 30 min), blocked in normal serum (Vectastain ABC Kit; Vector Lab., Inc., CA, USA), incubated with primary antibodies (solution with saline, 1:100) at 4 °C overnight, rinsed and incubated with secondary antibody (EliVision plus, DAB Kit, 9902).
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