SFMCs were seeded on coverslips and, at day 21, fixed in 4% formaldehyde and blocked in 5% donkey serum (32 (link)). Coverslips were stained with primary anti-human antibodies: anti-PD-1 (Abcam, Cambridge, UK), anti-PD-L1 (Beckman Coulter), and matching isotype controls in similar concentration. A secondary antibody was diluted 1:200: donkey anti-mouse 488 (Jackson ImmunoResearch,PA, USA). Coverslips were mounted with DAPI/anti-fade and examined in a confocal microscope (LSM710, Zeiss) equipped with Zen2009 software (Zeiss,Jena, Germany).
Anti pd 1
Manufactured by Abcam
Sourced in United States, China, United Kingdom
Anti-PD-1 is a laboratory reagent used in research applications. It is an antibody that binds to the PD-1 (Programmed Cell Death 1) protein, which is involved in the regulation of immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Anti pd l1, by Abcam (6 mentions)
Anti cd8, by Abcam (3 mentions)
Anti pd l1, by Cell Signaling Technology (3 mentions)
Anti cd4, by Abcam (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti cd3, by Abcam (2 mentions)
Lsm 710, by Zeiss (1 mentions)
Donkey anti mouse 488, by Jackson ImmunoResearch (1 mentions)
Zen 2009, by Zeiss (1 mentions)
Facscalibur, by BD (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rat, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti goat igg h l, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Anti gr 1, by BD (1 mentions)
Anti cd8, by BD (1 mentions)
Anti cd4, by BD (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Anti foxp3, by Novus Biologicals (1 mentions)
20 protocols using anti pd 1
1
Immunofluorescence Staining of PD-1 and PD-L1 in SFMCs
2
Isolation and Flow Cytometry of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, peripheral elbow venous blood (5 ml) was obtained from all patients within 24 h after admission. The samples were collected in tubes with heparin and added with 5 ml phosphate buffer (PBS) and then centrifuged at 1500 g for 10 min. The cell layer was then collected, added with 5 ml PBS, following with centrifugation at 1200 g for 5 min. After removing the supernatant and washing with PBS, the samples were further centrifuged at 1000 g for 2 min to obtain the peripheral blood mononuclear cells. The cells with density of 1 × 106/ml were maintained in RPMI-1640 Medium (Sigma-Aldrich, St. Louis, MO, USA).
For measurement of PD-1+ and LAG-3+ T cells, flow cytometry was performed as described elsewhere [17 (link), 18 (link)]. Antibodies used in this study included anti-CD4, anti-CD8, anti-PD-1 and anti-LAG-3 (all purchased from Abcam, USA). The measurement was conducted on a FACS Calibur flow cytometry analyzer (BD Biosciences) using Diva software (version 6.1, BD Pharmingen).
For measurement of PD-1+ and LAG-3+ T cells, flow cytometry was performed as described elsewhere [17 (link), 18 (link)]. Antibodies used in this study included anti-CD4, anti-CD8, anti-PD-1 and anti-LAG-3 (all purchased from Abcam, USA). The measurement was conducted on a FACS Calibur flow cytometry analyzer (BD Biosciences) using Diva software (version 6.1, BD Pharmingen).
3
Immunophenotyping of Early RA Synovial Biopsies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin-fixed paraffin-embedded early RA synovial biopsies were cut into 5μM sections and mounted on Fisherbrand Superfrost Plus Slides. The sections were stained with anti-CD3 (Cat# 103R-96, Cell Marque) and anti-PD1 (Cat# ab137132, Abcam) antibodies. PD-L1 analysis was performed using PD-L1 IHC 22C3 pharmDx following the manufacture instructions. Human tonsil and cell lines expressing PD-L1 (IHC 22C3 pharmDx control cell line slide) were used as positive controls for PD-L1 staining. The markers were visualized with 3,3'-diaminobenzidine and the relative expression of each marker was noted. CD3 and PD1 staining was reported as CD3+% or PD1+% cells within a total magnification of 100x across the entire biopsy section. PD-L1 staining was denoted as <1%, ≥1% or ≥50%, respectively.
4
Immunophenotyping of Tumor-Infiltrating Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen sections of resected tumors were cut into 8‐μm serial slices. Slices were fixed in 4% paraformaldehyde for 5 min at room temperature, followed by serum‐free blocking protein (Dako). Samples were subsequently stained with primary antibodies, such as anti‐CD4 (1:50; BD Pharmingen), anti‐CD8 (1:150; BD Pharmingen), anti‐PD‐1 (1:100; Abcam), anti‐Foxp3 (1:400; Novus Biologicals) and anti‐dendritic cell marker which reacts with dendritic cell inhibitory receptor 2 (1:50; Novus Biologicals), and anti‐Gr‐1 (1:100; BD Pharmingen) antibodies overnight at 4°C. After washing, fluorescence‐labeled secondary antibodies (Alexa Fluor 594 goat anti‐rat, Alexa Fluor 488 goat anti‐rabbit and Alexa Fluor 488 donkey anti‐goat IgG H + L [1:250, Life Technologies]) were then applied to the sections for 30 min at room temperature. DAPI (Vector Laboratory) was used for nuclear staining. Stained slides were finally imaged using an Olympus BX61 scanning fluorescence microscope. For quantification data, counting was performed in three random fields at ×200 magnification per tumor tissue specimen.
5
Immunohistochemical Evaluation of T-cell Infiltration and PD-1/PD-L1 Expression in Cervical Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical method was performed to detect the infiltration of T cells and the expressions of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) in the local cervix. Cervical tissue samples were fixed in 10% formalin. Immunohistochemistry was performed using our previously published protocol (15 (link)). In brief, tissue sections were incubated with one of the following primary antibodies: anti-CD3 (1 : 500; ZsBio, Beijing, China), anti-CD4 (1 : 500; Abways Technology, Shanghai, China), anti-CD8 (1 : 500; Abways Technology), anti-PD-1 (1 : 1000; Abcam, Cambridge, Massachusetts, USA) and anti-PD-L1 (1 : 1000; Abcam). Immunodetection was performed using an appropriate biotinylated immunoglobulin and a horseradish peroxidase-labeled avidin kit (ZsBio) with diaminobenzidine (DAB) as the substrate. Finally, the sections were lightly counterstained with hematoxylin for 30 s.
6
Co-culture of DLBCL and CD8+ T cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A double-chamber co-cultured system (Millipore) was employed for co-culture. The Transfected FARAGE or U2932 cells were seeded in the upper chamber whereas the CD8+ T cells were in the lower chamber, allowing the direct contact of DLBCL cells with immune cells. Anti PD-1 (Abcam) or anti-PD-L1 (Abcam) was added. CD8+ T cells were sorted by using a EasySep™ Human CD8+ T Cell Isolation Kit (STEMCELL,Vancouver, BC, Canada). The percentage of CD8+ T cells or CD8+ T cell apoptosis was detected by flow cytometry with the application of Annexin V-FITC Apoptosis Kit (Becton Dickinson, Franklin Lakes, NJ, USA) in reference to the manufacturer’s instructions50 (link),51 (link).
7
Tissue Microarray Analysis of HCC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue microarrays containing 33 HCC tissue cores (2 mm in diameter, Supplementary Table S1 ) were ordered from the Shanghai Outdo biotech company (HLiv-HCC180Sur-05). For immunofluorescence double staining, sections were first deparaffinized and then incubated with retrieval solution (ZSGB-Bio, Beijing, China) for 15 min in a microwave oven. After treatment with 10% goat serum at 37 °C for half an hour, the tissues were incubated with anti-CD56 (Abcam, Cambridge, MA, USA) and anti-PD-1(Abcam) antibodies overnight. The sections were then incubated with FITC or PE-labeled secondary antibodies (ZSJQ-Bio). 4',6-diamidino-2-phenylindole was used for counterstaining. The staining was detected on a Zeiss DM2500 microscope with × 20 objectives.
8
Western Blot Analysis of Immune Checkpoint Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were washed in PBS and immediately lysed using cell lysis buffer containing 1% Triton X-100, 1% NP40, 50 mM Tris-Cl pH 8, 150 mM NaCl, 2 mM EDTA, 1 mM Na3VO4, 1 mM NaF, phosphatase inhibitor cocktail (Sigma), and complete protease inhibitor cocktail (Roche). Protein concentration was quantified by BCA assay (Bio-Rad). Equal amounts of protein were resolved on an SDS-PAGE gel and then transferred onto nitrocellulose membranes (Millipore, Bedford, USA). After blocking in 5% skimmed milk for 30 min, the membranes were incubated with primary antibodies, such as anti-MAGT1 (Abcam, 1:1000), anti-PD-1 (Abcam, 1:1000), anti-NKG2D (Abcam, 1:1000), anti-β-actin (Abcam, 1:1000), and anti-GAPDH (CST, 1:1000), at 4 °C overnight. After washing, the membrane was incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, USA; 1:1000) at RT for 1 h. The signal was detected using enhanced chemiluminescence system (Millipore). The band intensity was analyzed with Image J software (NIH, USA) and the protein levels were normalized by GAPDH or β-actin.
9
Immunohistochemical Profiling of LUAD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LUAD tissues were fixed with 10% formalin and embedded in paraffin. Then, the tissues were cut into 5-μm-thick sections and incubated overnight with primary antibodies anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD20, anti-PD1 (Abcam,UK). The sections were subsequently incubated with a secondary antibody (Abcam,UK) at 37 °C for 1.5 h and stained with a 3,3-diaminobenzidine solution.
10
PD-1/PD-L1 Expression in Predicting PTCL Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To define predictors of clinical response to PD-1 blockade in patients with PTCL, we compared PD-1/PD-L1 expression by immunohistochemistry (IHC) in patients who achieved a response to nivolumab, in those without a response and in patients with HPD. Paraffin embedded tissue of pre-treatment samples were deparaffinized in xylene. After cleared through graded ethanol series, endogenous peroxidase was quenched by incubation in 50% methanol/H2O2. The sections were pretreated 30 min with 50 mM EDTA, pH 8.0 using a steamer and cooled for an additional 5 min. The staining was performed using anti-PD-1 (Abcam, 1:50) or anti-PD-L1 (Abcam, 1:50). The sections were stained with hematoxylin and rinsed well in tap water. The slides were observed and captured images with MOTIC EasyScan.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!