Formalin-fixed, paraffin-embedded slides were deparaffinized and sequentially rehydrated using ethanol. The slides were immersed in antigen retrieval solution (Dako, Santa Clara, CA) and incubated for antigen retrieval at high pressure using a cooker. After cooking, the slides were incubated with 2.5% horse serum in phosphate-buffered saline (PBS) for blocking and then incubated overnight at 4°C with the primary antibodies. Mouse anti–SARS-CoV-2 nucleocapsid (Sino Biological, Beijing, China), rabbit anti-CD11c (Cell Signaling Technology), rabbit anti-CD19 (Cell Signaling Technology), rat anti-CD3 (Abcam), rat anti-MHCII (eBioscience), rabbit anti-podoplanin (PDPN; Santa Cruz Biotechnology, Dallas, TX), goat anti–surfactant protein C (SP-C; Santa Cruz Biotechnology), rabbit anti-CD31 (Abcam), and rabbit anti–transmembrane protein 173 (TMEM173; Proteintech, Rosemont, IL) were used as primary antibodies. Each primary antibody derived from diverse species was detected with the use of Alexa488-conjugated, Alexa568-conjugated, and Alexa647-conjugated secondary antibodies (secondary antibodies all from Invitrogen, Waltham, MA). Nuclear staining was performed with the use of DAPI (Abbkine, Wuhan, China). Confocal images were taken with a confocal microscope (Ts2; Nikon, Tokyo, Japan) of Kangwon Center for System Imaging.
Rat anti mhcii
Manufactured by Thermo Fisher Scientific
The Rat anti-MHCII is a laboratory reagent used for the detection and analysis of major histocompatibility complex class II (MHCII) molecules. MHCII molecules are cell surface glycoproteins that play a crucial role in the adaptive immune response by presenting antigenic peptides to T helper cells. The Rat anti-MHCII antibody can be used in various immunological techniques, such as flow cytometry, immunohistochemistry, and Western blotting, to identify and quantify MHCII-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Antigen retrieval solution, by Agilent Technologies (1 mentions)
Alexa 647 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cd31, by Abcam (1 mentions)
Rabbit anti cd11c, by Cell Signaling Technology (1 mentions)
Rat anti cd3, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abbkine (1 mentions)
Complete mini edta free cocktail, by Roche (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Human fibronectin, by Merck Group (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Rabbit anti her2, by Cell Signaling Technology (1 mentions)
Rat anti lamp1, by Abcam (1 mentions)
Texas red x, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Mouse anti gapdh, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Merck Group (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using rat anti mhcii
1
Multicolor Immunofluorescence Staining of SARS-CoV-2 Antigens
2
Protein Isolation and Western Blot Analysis
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Total proteins from mouse ventral midbrain, striatum and cerebellum samples were isolated in NP‐40 buffer (20 mM Tris–HCl pH 8; 137 mM NaCl; 10% glycerol; 1% NP‐40; 2 mM EDTA and protease inhibitors (cOmplete Mini EDTA‐free cocktail, Roche)) 1:20 (wt/vol). Protein concentration was determined using a bicinchoninic acid kit (Pierce, Rockford, IL). After boiling in Laemmli's buffer, 20 µg of protein was separated by electrophoresis on a 12% sodium dodecyl sulphate–polyacrylamide gel, transferred to nitrocellulose membrane, and blocked with 2% BSA, 5% or 2% non‐fat dried milk in PBS containing 0.05% Tween‐20 (vol/vol). Overnight incubation with primary antibody at 4°C followed. Primary antibodies were rat anti‐MHC II (1:750; eBioscience), mouse anti‐interferon‐γ (1:500; ThermoScientific, Cambridge, UK), rabbit anti‐tumour necrosis factor‐α (1:200; Abcam, Cambridge, UK), rabbit anti‐interleukin‐1β (1:500; Abcam), and mouse anti‐β‐actin (1:25,000; SigmaAldrich). Blots were then washed a second time in PBS‐Tween (0.05%) and incubated with an appropriate secondary antibody (Jackson Immuno Research). Blots were washed in PBS‐Tween (0.05%) and developed using the Supersignal West Dura kit (Pierce) as per manufacturer's instructions. Bands were visualized with an AlphaInnotech digital imaging system (San Leandro, CA) and quantified with AlphaEase FC 5.02 software.
3
Immunofluorescence Imaging of HER2, LAMP1, and MHC-II
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293T, E0771, and JAWSII cells were grown on 8-well slides (MatTek) coated with 1 µg/mL human fibronectin (EMD Millipore). Transfection was performed using Lipofectamine3000 (Thermofisher). For Lysotracker (ThermoFisher) staining, cells were incubated with 75 nm Lysotracker for 1 hour at 37°C. For endoplasmic reticulum (ER) staining, cells were incubated with 2 μL ER CellLight ER-RFP Bacman V.2.0 (Thermofisher) for 24 hours. Slides were washed with phosphate buffered saline (PBS), fixed with 10% formalin for 20 min and mounted with DAPI Fluoromount-G (SouthernBiotech). Staining was performed in 0.1% PBST with rabbit anti-HER2 (1:200, Cell Signaling), rat anti-LAMP1 (1:200, Abcam) or rat anti-MHCII (1:200, Thermofisher) at 4° overnight and antirabbit Alexa Fluor 488 (1:1000 dilution, Invitrogen) or antirat Texas Red-X (1:1000, invitrogen) at room temperature for 1 hour. Slides were mounted with Fluoromount-G with DAPI (Southern Biotech).
4
Western Blot Analysis of Immune Markers
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Collected LLC cells or tumor tissues were lysed and homogenized in RIPA lysis supplemented with protein inhibitor. Total proteins were quantified using a BCA assay kit (Beyotime, Shanghai, China). Then equal amounts of protein from each group were loaded and separated by SDS-PAGE followed by transferring to PVDF membranes (EMD Millipore Corp., Burlington, MA). After blocking with 5% bovine serum albumin (BSA, Sigma) for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4°C. The next day, membranes were incubated with the corresponding HRP-conjugated secondary antibodies (1:2000, Sigma) for 2 h at room temperature. Finally, bands were visualized using the ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA). Primary antibodies used were rabbit anti-CD91 (1:2000), mouse anti-GAPDH (1:1000), rabbit anti-CD206 (1:2500) and rat anti-MHC II (1:1000), all of which were purchased from Thermo Fisher Scientific. GAPDH served as the internal control.
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