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.
Anti foxp3
Manufactured by Novus Biologicals
Sourced in Germany
Anti-FoxP3 is a laboratory product used to detect the FoxP3 protein, which is a transcription factor involved in the development and function of regulatory T cells. The product is designed for research use only and its core function is to enable the identification and study of FoxP3-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
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 pd 1, by Abcam (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dako fluorescence mounting medium, by Agilent Technologies (1 mentions)
Zen 3.1 blue edition software, by Zeiss (1 mentions)
Lsm 510 confocal laser scanning microscope, by Zeiss (1 mentions)
Anti phospho nf κb p65 ser536, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg hrp, by Abcam (1 mentions)
Anti phospho iκbα ser32, by Cell Signaling Technology (1 mentions)
Ml120b, by Merck Group (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Goat anti rabbit igg hrp, by Abcam (1 mentions)
Goat anti mouse igg fitc, by Santa Cruz Biotechnology (1 mentions)
5 protocols using anti foxp3
1
Immunophenotyping of Tumor-Infiltrating Immune Cells
2
Immunostaining of Mouse RPE/Choroid Flatmounts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPE/choroid flatmounts from mouse eyes were prepared as previously described [75 (link)]. In short, eyes were enucleated, fixed in 4% paraformaldehyde for 12 min at room temperature, and sectioned at the limbus; the retinas were removed from the RPE/choroid/sclera and 6–8 radial sections were made. After incubation of the RPE/choroid/sclera tissue in 5% Triton X-100 in TBS overnight at 4 °C, the samples were incubated in blocking buffer for 1 h (5% BSA in TBS). Subsequently, an incubation with rabbit polyclonal anti-FoxP3 (Novus, 1:200) antibody and ActiStain555-conjugated phalloidin (Biomol, Hamburg, Germany, 1:500) followed for 48 h. Rat anti-mouse CD102 (BD-Pharmingen, Heidelberg, Germany, 1:200) was used to visualize the laser scars. After a few washes, the samples were incubated for 1 h at room temperature with the appropriate Alexa Fluor®-conjugated secondary antibodies (Thermo Fisher Scientific; 1:1000). Tissues were embedded in DAKO fluorescence mounting medium (Agilent, Ratingen, Germany) and images were taken using a LSM 510 confocal laser-scanning microscope (Zeiss, Jena, Germany) and digitalized using ZEN 3.1 Blue Edition software (Zeiss, Oberkochen, Germany).
3
Investigating CCL28-Mediated Immune Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibodies and reagents were obtained from the following sources: anti-CCL28 (Santa Cruz, sc-376654), anti-GAPDH (Abcam, ab9485), anti-phospho-NF-κB p65 (Ser536) (Cell Signaling, 3033), anti-NF-κB p65 (Cell Signaling, 8242), anti-phospho-IκBα (Ser32) (Cell Signaling, 2859), anti-IκBα (Cell Signaling, 4812), anti-FoxP3 (Novus, NB100-39002), anti-CCR10 (Invitrogen, PA1-21617), ML120B (Sigma, SML1174). Goat anti-rabbit IgG-HRP (Abcam, ab6721), goat anti-mouse IgG-HRP (Abcam, ab6789), goat anti-rabbit IgG-PE (Santa Cruz, sc-3739), goat anti-mouse IgG-FITC (Santa Cruz, sc-2010), goat anti-mouse peroxidase-conjugated IgG (Millipore, AP124P). Neutralizing antibodies against mouse IL-1β (AF401), TNF-α (AF410), CCL28 (MAB533), CCR10 (MAB2815), CCR3 (MAB1551), CD25 (AF2438) and IgG isotype control (43414), and recombinant mouse CCL28 (533-VI) were purchased from R&D Systems.
4
Immunohistochemical Analysis of Mouse Bone
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At the end of experiments, mouse long bones were harvested and fixed in 10% neutral buffered formalin for 24 hours followed by decalcification in Immunocal (StatLab, McKinney, TX) for 3 days. Tissues were then processed, embedded into paraffin, and sectioned 5 mm thick. For immunohistochemistry, sections were de-paraffinized and rehydrated using xylene followed by ethanol gradient. Antigen retrieval was performed by incubating samples at 60 degrees celsius in Citrate buffer (pH 6.0) followed by quenching of endogenous peroxidase activity with 3% H2O2. Sections were blocked using DAKO solution with background reducing components. Sections were incubated overnight with a 1:200 dilution of anti-Luciferase (Novus), anti-Nrp1 (Novus) or anti-FoxP3 (Novus) antibody. Sections were rinsed in phosphate-buffered saline (PBS) followed by a 1:1000 dilution of biotinylated secondary antibody for one hour. Post-secondary antibody incubation, the sections were incubated with stereptavidin-HRP (2 ug/ml) for 20 min. After extensive washing with PBS, sections were developed using Impact DAB kit (Vector Biolabs).
5
Immunohistochemical Analysis of Splenic Immune Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin sections were stained with hematoxylin and eosin and periodic acid-Schiff and evaluated by a nephropathologist using Banff criteria. 14 Three micrometer formalin-fixed paraffin-embedded spleen sections were stained with anti-rat IgG-AlexaFluor647 (Thermo Fisher A21472, Waltham) for plasmablasts/plasma cells (PB/PC), anti-CD20 (Santa Cruz sc-393894) for B-cell follicles, anti-Ki67 (eBioscience, 11-5698) for proliferating GC cells, anti-CD3 (Abcam, 5690) for T follicular helper (TfH), and anti-foxp3 (Novus Bio, NB100-39002) for T follicular regulatory cells (Tfr), which were counted within Ki67 + GC regions. Staining was performed according to a standard immunohistochemistry protocol. 15 Cells were quantified using Histoquest software.
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!