Ten micrograms of proteins of each group were separated by 11 % SDS-PAGE and transferred onto nitrocellulose membranes. The blots were incubated with primary antibodies against human Nrf2(ab137550, Abcam. 1:400), TRAP-1 (ab2721, Abcam. 1:700), α-SMA (ab108424, Abcam. 1:500), FAP (ab227703, Abcam. 1:600), Collagen I (ab34710, Abcam.1:400), HDAC1 (ab7208, Abcam. 1:500), p-Nrf2 (ab76026, Abcam. 1:300) and β-actin (ab6276, Abcam. 1:2000) overnight at 4 °C, following incubated with the corresponding HRP-conjugated secondary antibody (ab205718, Abcam.1:3500). After washing the membranes twice with Tris Buffered Saline with Tween (TBST, pH = 7.4) and the bands in the membranes were visualized by enhanced chemiluminescence (ECL, Pierce, USA). After incubation for 2 min, the membranes were exposed to x-ray films. The protein bands were scanned and analyzed. The levels of these proteins were calculated as the ratio of the intensity of the specified protein to that of β-actin by using ImageJ2x software (National Institutes of Health, Maryland, USA). The intranuclear Nrf2 protein levels were calculated as the optical density ratio of intranuclear Nrf2 to HDAC1, and the phosphorylated Nrf2 was calculated as optical density ratio of phosphorylated Nrf2 to its total.
Ab227703
Manufactured by Abcam
Sourced in United Kingdom
Ab227703 is a laboratory equipment product. It is a piece of lab equipment used for a specific function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab34710, by Abcam (2 mentions)
Ab76026, by Abcam (2 mentions)
Ab2721, by Abcam (2 mentions)
Ab7208, by Abcam (2 mentions)
Ab108424, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab137550, by Abcam (2 mentions)
Ab6276, by Abcam (2 mentions)
Wizard 2480, by PerkinElmer (1 mentions)
Bond rx autostainer, by Leica (1 mentions)
Aperio at2, by Leica (1 mentions)
Buffered formalin, by Thermo Fisher Scientific (1 mentions)
Bond polymer refine detection, by Leica (1 mentions)
Ab68153, by Abcam (1 mentions)
Ab32576, by Abcam (1 mentions)
Ab80590, by Abcam (1 mentions)
Ab652, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Bx51 fluorescence microscope, by Olympus (1 mentions)
Dp72 camera, by Olympus (1 mentions)
8 protocols using ab227703
1
Western Blot Analysis of Cellular Proteins
2
Ex Vivo Biodistribution of Radiolabeled Probes
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After imaging collection and image reconstruction, samples from euthanized experimental mice, including blood and multiple significant organs, were harvested and weighed. The radioactivity in each collected tissue was counted using an automated gamma counter (Wizard 2480, PerkinElmer) and the accumulation of radioactivity in different tissues and organs was quantified and expressed as %ID/g (mean ± SD). [177Lu]Lu-DOTA-FD1 (~1.11 MBq, n = 3), [177Lu]Lu-DOTA-FD2 (~1.11 MBq, n = 3), and [177Lu]Lu-DOTA-FD3 (~1.11 MBq, n = 3) were injected into the tail vein of free nude mice at 6 h, 24 h, and 24 h for ex vivo biodistribution to verify the circulation time of the 3 probes in vivo. Biodistribution data from tumor-bearing mice injected with [177Lu]Lu-DOTA-FD3 at 45 h (~1.11 MBq, n = 4) and 90 h (~1.11 MBq, n = 3) validated the prolonged enrichment of the probe at the tumor site. At the end of each probe imaging and radioactivity counting study, tumor tissues were fixed in paraformaldehyde tissue fixative. Briefly, sections of 10 μm were cut and stained for H&E following the standard protocols. IHC staining with a FAP antibody (ab227703, Abcam) was performed only on tumor tissues following accepted techniques.
3
Immunoprofiling of Mouse Tumor Samples
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Mouse tumors were fixed in 10% buffered formalin (Thermo Fisher Scientific) overnight and moved to 70% ethanol thereafter. Paraffin embedding, tissue sectioning, H&E staining, Immunohistochemistry (IHC) and Trichrome staining was performed by HistoWiz Inc. (histowiz.com) using a Standard Operating Procedure and fully automated workflow. Samples were processed, embedded in paraffin, and sectioned at 4μm. Immunohistochemistry was performed on a Bond Rx autostainer (Leica Biosystems) with Heat-Induced Epitope Retrieval (pH6 or pH9) using standard protocols. The Bond Polymer Refine Detection (Leica Biosystems) was used according to the manufacturer’s protocol. After staining, sections were dehydrated and film coverslipped using a TissueTek-Prisma and Coverslipper (Sakura). Whole slide scanning (40x) was performed on an Aperio AT2 (Leica Biosystems). For IHC antibodies used were: α-CD8 (#LSB3914, LSBio, 1:600) and α-FAP (#ab227703, Abcam, 1:100).
4
Immunohistochemical Analysis of Tumor Samples
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Tumor samples were harvested and fixed using formalin, followed by paraffin-embedment. Sections were prepared into sections (5-µm thickness) for staining protocol. Sections were first dewaxed in xylene and rehydrated with serial ethanol gradients and washed in deionized water. Subsequently, sections were blocked in PBS with 1% BSA, 1% donkey serum and 0.3% Triton X-100 and 0.01% sodium azide for 1 h, room temperature. After blocking, the slides were exposed to MEK (1:200, Abcam, ab32576), STAT3 (1:200, Abcam, ab68153), PDCD4 (1:200, Abcam, ab80590), and FAP antibody (1:200, Abcam, ab227703) at 4 °C overnight, washed and incubated in biotinylated link universal antiserum for 1 h at room temperature. Primary antibodies were diluted in blocking buffer and incubated in the cold overnight. The next day, sections were washed in PBST 3 times and incubated with secondary antibodies in blocking buffer (1 h, room temperature). Stained sections were then observed under a microscope and micrographed using a mounted digital camera.
5
Immunohistochemical Evaluation of FAP and GLUT1
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Immunohistochemistry was performed on formalin-fixed paraffin-embedded human tissue samples according to the standard laboratory procedures (17 (link)). The following antibodies were used: anti-GLUT1 Abcam ab652 (RRID:AB 305540), diluted 1:5,000; anti-FAP α-antibody (SP325); and Abcam ab227703, diluted 1:100. Immunohistochemical expression was evaluated on tumor cells and tumor stroma, and the percentage of intratumoral necrosis related to the tumor areas was also assessed. A simplified visual FAP/GLUT1 grading was applied for stromal and tumor cells, as well as for necrosis. A FAP/GLUT1 grading legend is shown in Table 1 . For larger neoplasms, a central slice of the tumor was stained completely. Immunohistochemical analyses were performed on a resection of bioptic samples of the primary or local-recurrence tumors before 68Ga-FAPI-46 or 18F-FDG PET/CT and consequently do not correspond to visible PET lesions. Two pathologists and 2 biologists performed masked immunohistochemical analysis in consensus.
6
Immunohistochemical Staining of FAP in CAFs
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Routine IHC method was performed for the staining of FAP. The primary antibody was rabbit anti-human monoclonal antibody against FAP (ab227703, Abcam, UK, dilution 1/200), with corresponding horseradish peroxidase (HRP) conjugated secondary antibody (ab6721, Abcam, UK, dilution 1/200). The FAP positive CAFs were indicated by both morphological features and the IHC reaction results. The reaction products were visualized with diaminobenzidine (DAB, DAKO, Denmark). Then the slides were evaluated by two senior pathologists, who were blinded to the patients’ clinical features and outcomes. A consensus was achieved using a multi-headed microscope in case of discrepancy. In brief, at least 4 standard-compliant vision fields of FAP expression (magnification, × 200) per patient was considered to be adequate, with no focus on hotspots. The digital images were captured under Olympus BX51 fluorescence microscope equipped with Olympus DP72 camera (Olympus Optical Co., Ltd., Tokyo, Japan). Identical settings were used for every photograph, so as to minimize the selection bias.
7
FAP-α Antibody Sourcing Protocol
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Water soluble phthalocyanine dye IR700 was obtained from Li‐Cor Bioscience. Anti‐FAP‐α polyclonal sheep Ab, AF3715, monoclonal mouse Ab MAB3715, and monoclonal rat Ab MAB9729 were purchased from R&D Systems. Anti‐FAP‐α monoclonal mouse Ab BMS168 was purchased from eBioscience. Anti‐FAP‐α Abs, ab137549, ab218164, ab28244, ab53066, ab207178, and ab227703, were purchased from Abcam. Anti‐FAP‐α polyclonal rabbit Ab PA5‐51057 and sheep IgG isotype control (Cat. No. 31243) were purchased from Thermo Fisher Scientific.
8
Western Blot Analysis of Cellular Proteins
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Ten micrograms of proteins of each group were separated by 11 % SDS-PAGE and transferred onto nitrocellulose membranes. The blots were incubated with primary antibodies against human Nrf2(ab137550, Abcam. 1:400), TRAP-1 (ab2721, Abcam. 1:700), α-SMA (ab108424, Abcam. 1:500), FAP (ab227703, Abcam. 1:600), Collagen I (ab34710, Abcam.1:400), HDAC1 (ab7208, Abcam. 1:500), p-Nrf2 (ab76026, Abcam. 1:300) and β-actin (ab6276, Abcam. 1:2000) overnight at 4 °C, following incubated with the corresponding HRP-conjugated secondary antibody (ab205718, Abcam.1:3500). After washing the membranes twice with Tris Buffered Saline with Tween (TBST, pH = 7.4) and the bands in the membranes were visualized by enhanced chemiluminescence (ECL, Pierce, USA). After incubation for 2 min, the membranes were exposed to x-ray films. The protein bands were scanned and analyzed. The levels of these proteins were calculated as the ratio of the intensity of the specified protein to that of β-actin by using ImageJ2x software (National Institutes of Health, Maryland, USA). The intranuclear Nrf2 protein levels were calculated as the optical density ratio of intranuclear Nrf2 to HDAC1, and the phosphorylated Nrf2 was calculated as optical density ratio of phosphorylated Nrf2 to its total.
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