The largest database of trusted experimental protocols

3 protocols using anti cd4

1

Plasmid Transfection and Immunoblot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Plasmid transfections were performed using Lipofectamine 2000 (Invitrogen) as per the manufacturer's protocol. Relative levels of different proteins were compared by immunoblot analysis. Respective empty vectors were used to normalize transfection. After 48 h of transfection, cells were lysed in RIPA lysis buffer (Cell Signaling Technology), and protein estimation was done using the BCA Protein Estimation Kit (Pierce Biotechnology, Inc.). The primary antibodies used were anti-Myc (Clontech), anti-glyceraldehyde 3-phosphate dehydrogenase (GADPH) (Cell Signaling Technology), and anti-CD4 (Novus Biologicals). The secondary antibodies used were anti-rabbit/mouse-horse radish peroxidase conjugated (Jackson ImmunoResearch). The proteins of interest were detected with EZ western horseradish peroxidase substrate (Biological Industries, Israel). GAPDH was used as a loading control in all cases.
+ Open protocol
+ Expand
2

Immunofluorescence Analysis of Immune Markers in Gastric Cancer Mucosa

Check if the same lab product or an alternative is used in the 5 most similar protocols
Mucosa from GC patients was fixed in 10% formalin and embedded in paraffin. Paraffin-embedded sections were probed with anti-CD4+ (Novus Biologicals, Littleton, CO, USA), anti-CD8+ (Novus Biologicals), anti-PD-L1 (Invitrogen, Carlsbad, CA, USA), and anti-CTLA-4 (Invitrogen) primary antibodies at 4°C overnight. They were then stained with secondary antibodies conjugated with fluorescein isothiocyanate (Santa Cruz Biotechnology, Santa Cruz, CA, USA), allophycocyanin (Invitrogen), and phycoerythrin (Southern Biotech, Birmingham, AL, USA) at room temperature for 2 h. Nuclei were stained with 4,’6-diamidino-2-phenylindole (DAPI; Invitrogen). Immunofluorescence images were obtained using an LSM 700 confocal microscope (Zeiss, Oberkochen, Germany) at 200× magnification. Images were analyzed using ZEN 2 (blue edition) (Zeiss).
+ Open protocol
+ Expand
3

Immunohistochemical Analysis of Tumor-Infiltrating Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
SW-1 bearing mice were euthanized 7 days after their tumors had been injected once with the 3 mAb injection, and the tumors were fixed in 10% paraformaldehyde. Four-micron sections of formalin-fixed paraffin-embedded tissue were cut and placed on Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA). The sections were deparaffinized and rehydrated through graded alcohols. Antigen retrieval was carried out with Target Retrieval Solution (DAKO Corporation; Carpinteria, CA) in a microwave for 10 minutes. Endogenous peroxidase activity was blocked with 3% H2O2. The slides were washed and incubated with anti-CD4 (Novus biologicals, Littleton, CO), anti-CD8 (Biorbyt, San Francisco, CA) or anti-CD19 (Biorbyt, San Francisco, CA) mAbs overnight at 4°C, after which they were washed and incubated with Anti-Rabbit IgG-Peroxidase antibody (Sigma-Aldrich, St.Louis, MO). Color development was accomplished by incubation in diaminobenzidine (DAKO Corporation; Carpinteria, CA). The slides were counterstained by hematoxylin (DAKO Corporation) and coverslipped with permanent mounting media.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!