Immunostaining was performed as previously described to visualize perforin expression in mouse lung tissues (25 (link)). Rat anti-mouse perforin (abcam Ab16074) and goat anti-rat Alexa 594 were used as primary and secondary antibodies, respectively. Images were acquired with a Carl Zeiss LSM 780 system.
Ab16074
Manufactured by Abcam
Sourced in United Kingdom
Ab16074 is a primary antibody that targets the Glial fibrillary acidic protein (GFAP) in human, mouse, and rat samples. GFAP is an intermediate filament protein that is expressed in astrocytes and ependymal cells in the central nervous system.
Automatically generated - may contain errors
Lab products found in correlation
Lsm780 system, by Zeiss (1 mentions)
Ab4059, by Abcam (1 mentions)
Ab9657, by Abcam (1 mentions)
Ab18256, by Abcam (1 mentions)
Aperio imagescope software, by Leica (1 mentions)
Hematoxylin, by Bio-Optica (1 mentions)
Ab828, by Abcam (1 mentions)
Dmrd optical microscope, by Leica (1 mentions)
Vulcan fast red chromogen, by Biocare Medical (1 mentions)
Alkaline phosphatase conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Ab15580, by Abcam (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Vectastain elite abc hrp system, by Vector Laboratories (1 mentions)
L9393, by Merck Group (1 mentions)
Lsm880 confocal microscope system, by Zeiss (1 mentions)
4 protocols using ab16074
1
Visualizing Perforin Expression in Mouse Lungs
2
IRIN Silicasome Tumor Therapy Evaluation
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Tumor‐bearing mice received IV injection to deliver an IRIN dose of 40 mg kg−1 (free IRIN or IRIN silicasome) per injection every 3 days for a total of 3 administrations. Control animals received saline only. Animals were sacrificed 72 h after the last injection. To confirm the impact on tumor growth, ex vivo bioluminescence imaging was performed to assess image intensity at the primary and metastatic tumor sites. Primary tumors were fixed in 10% formalin, followed by paraffin embedding and sectioning to provide 4 µm slices for IHC analysis in the UCLA Translational Pathology Core Laboratory (TPCL). The slides were scanned and images were assessed by using Aperio ImageScope software (Leica). Primary antibodies to CD8 (#14‐0808‐82) and FoxP3 (#13‐5773‐82) were purchased from ThermoFisher; CRT (ab2907), while antibodies to HMGB1 (ab18256), granzyme B (ab4059), perforin (ab16074) and IFN‐γ (ab9657) were purchased from Abcam. The antibody to LC‐3 (#0231‐100/LC3‐5F10) was purchased from Nanotools, while the antibody to PD‐L1 (#64988) was purchased from Cell Signaling Technology.
3
Immunohistochemical Analysis of Mammary Tumors
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Whole mounts were prepared as previously described [15 (link)]. Mammary tumors were fixed in formalin and embedded in paraffin. hematoxylin and eosin staining (H&E) was used to assess histology. Infiltration by CD3+ and perforin-positive cells was determined through immunohistochemistry with anti-CD3 (Ab828; Abcam, Cambridge, UK) and anti-perforin (Ab16074, Abcam) antibodies as previously described [16 (link)]. Immunostaining was developed using alkaline phosphatase-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA, USA) and vulcan fast red chromogen (Biocare Medical, Concord, CA, USA). Slides were counterstained with hematoxylin (BioOptica, Milan, Italy) and images were acquired by Leica DMRD optical microscope (Leica, Milton Keynes, UK).
4
Immunohistochemical Analysis of Tissue Sections
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Heat-induced antigen-retrieved tissue sections were subjected to Vectastain Elite ABC-HRP system (PK6101, Vector Labs) for immunohistochemistry according to the manufacturer’s instructions. Primary antibodies were as follows: anti-Ki67 (1/10000; ab15580, Abcam), anti-Laminin (1/750; L9393, Sigma-Aldrich), anti-Perforin (1/200; ab16074, Abcam), and anti-PIBF1 (1/500; PMID:1463410734 (link)). In the case of staining Perforin, peroxidase-conjugated donkey anti-rat IgG (712-035-150, Jackson ImmunoResearch Laboratories) was applied as a secondary antibody. Labyrinthine Ki67-positive trophoblast cells or decidual Perforin-positive cells were counted in three randomly selected ×40 fields of each sample.
For immunofluorescence staining, antigen-retrieved tissue sections were subjected to primary antibodies, subsequently captured by Alexa Fluor-conjugated secondary antibodies. In vitro samples, including fixed cells and hHO cryosections, were permeabilized with 0.1% Triton X-100 and subjected to antibodies. The sections were mounted with DAPI (D1306, Molecular Probes), and examined under Zeiss LSM 880 confocal microscope system (Carl Zeiss, Jena, Germany). CD31, cTnT, or PDGFRβ-stained areas were quantified using ImageJ software (NIH). The antibodies used for immunofluorescence staining are listed in Supplementary Data6 .
For immunofluorescence staining, antigen-retrieved tissue sections were subjected to primary antibodies, subsequently captured by Alexa Fluor-conjugated secondary antibodies. In vitro samples, including fixed cells and hHO cryosections, were permeabilized with 0.1% Triton X-100 and subjected to antibodies. The sections were mounted with DAPI (D1306, Molecular Probes), and examined under Zeiss LSM 880 confocal microscope system (Carl Zeiss, Jena, Germany). CD31, cTnT, or PDGFRβ-stained areas were quantified using ImageJ software (NIH). The antibodies used for immunofluorescence staining are listed in Supplementary Data
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