For immunofluorescence studies, tumors were fixed for 24 h in 4% paraformaldehyde (PFA), followed by overnight incubation in a 30% sucrose solution. Tumors were then snap-frozen in optimal cutting temperature compound (OCT) and 5 μm sections were obtained. For CD8 (Cell Signaling, Danvers, MA, USA, 98941) and CD11c (Cell Signaling, 97585S) staining, slides were fixed for 10 min in 4% PFA before antigen retrieval in citrate buffer (pH 5.7) with 0.5% Triton. After the addition of an AlexaFluor-488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA, A11034), slides were scanned (3DHistech Pannoramic P250 Flash III, Budapest, Hungary, 20X) and the extent of CD8 and CD11c staining was quantified over the total tumor area using Visiopharm software. For TIL analysis by flow cytometry, tumors were mechanically dissociated and single cell suspension was obtained by passing them through a 70 µm strainer. Cells were stained with CD45-BV510 (BD Pharm, 513151), CD3-APC (BD Pharm, 553066), CD8a-FITC (BD Pharm, 553031) and CD4-BV421 (BD Pharm, 553066) for 15 min RT.
Pannoramic p250 flash 3
Manufactured by 3DHISTECH
Sourced in Hungary
The Pannoramic P250 Flash III is a high-speed digital slide scanner designed for use in pathology laboratories. It is capable of scanning whole slide images at a high resolution and speed, enabling efficient digitization of tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Caseviewer software, by 3DHISTECH (2 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Cd3 apc, by BD (1 mentions)
Cd4 bv421, by BD (1 mentions)
Cd45 bv510, by BD (1 mentions)
Cd8a fitc, by BD (1 mentions)
Ab64238, by Abcam (1 mentions)
Ht501128 4l, by Merck Group (1 mentions)
Halo software, by Indica Labs (1 mentions)
Cell observer spinning disk confocal microscope, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
4 protocols using pannoramic p250 flash 3
1
Immunofluorescence and Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes
2
Muscle Development and Fiber Analysis
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Quadriceps and gastrocnemius muscles were dissected at 2.5 or 5 months of age and weighed. They were immersion-fixed in 4% PFA for 72 hours, paraffin-embedded and sectioned transversely at 20 micrometers. Sections were deparaffinized in Toluene 100% for 3x5 minutes, immersed in isopropanol 100% for 30 seconds and then rehydrated in a reverse ethanol series (100 to 30%, 2 min for each step). Sections were histologically stained with hemalun/eosin or labeled with Wheat Germ Agglutinine (WGA)/rhodamine (rabbit; 1:150; Vector RL-1022; Laboconsult) diluted in PBS and applied for 2 hours at RT. After washes in PBS, the slides were mounted as described above. Images were acquired with a Slide Scanner (3DHistech Pannoramic P250 Flash III) using CaseViewer software (3DHistech Ltd.) software. Central nuclei were counted on the entire RF portion of the quadriceps on at least 3 sections for each genotype (n = 3). For each muscle (n = 3), muscle fiber area was measured for at least 50 fibers in 5 sections using ImageJ software Raw data were exported to Microsoft Excel to draw the histograms.
3
Immunohistochemical Analysis of Pancreatic Tissue
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Dissected pancreata were fixed in 4% paraformaldehyde (PFA; HT501128-4L, Sigma Aldrich, Overijse, Belgium) for 4 h at 4 °C, before paraffin embedding. Sections of 6 µm were deparaffinized and antigen retrieval was performed using citrate (pH 6.0) or Tris-EDTA (pH 9.0) buffer in Lab Vision PT Module (Thermo Fisher Scientific, Merelbeke, Belgium). After permeabilization with PBS/0.3% Triton-100X (3051.4, Carl Roth, Karlsruhe, Germany) for 5 min, sections were blocked with solution 1 (3% low-fat milk, 5% BSA, 0.3% Triton-100X in PBS) for 45 min, at RT. Primary antibodies (Table 1 ) diluted in solution 1 were added and incubated overnight, at 4 °C. The next day, slides were washed with PBS/0.1% Triton-100X and incubated with secondary antibodies diluted in solution 2 (10% BSA, 0.3% Triton-100X) at 37 °C, for 1 h, before DAB staining (ab64238, Abcam, UK, Cambridge). Pictures were selected after scanning on Pannoramic P250 Flash III (3DHistech, Budapest, Hungary). Edematous areas were measured on hematoxylin and eosin (H&E)-stained sections (measurements were performed on the whole pancreas area) using the CaseViewer software (3DHistech, Budapest, Hungary). The quantification of CD45, NF-κB(p65) and P-ERKT202/Y204 staining was performed on whole pancreas using the HALO software (Indica Labs, Albuquerque, NM, USA).
4
Immunohistochemistry Protocol for Paraffin Sections
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Dissected tissues were fixed in 4% paraformaldehyde for 4 hours at 4 C, with gentle rotation, before paraffin embedding. Six-micronthick sections were first deparaffinized. Then antigen retrieval was performed using citrate (pH 6.0) or Tris-EDTA (pH 9.0) buffers using microwave (MW), Lab Vision PT Module (Thermo Fisher Scientific), or pressure cooker (CC; Supplementary Table S1). Sections were washed once with PBS and then permeabilized with PBS/0.3% Triton-100X for 5 minutes, at room temperature. Sections were blocked with solution 1 (3% low-fat milk, 5% BSA, 0.3% Triton-100X in PBS) for 45 minutes, at room temperature. Primary antibodies (Supplementary Table S1) diluted in solution 1 were added and incubated overnight, at 4 C. On the next day, slides were washed with PBS/0.1% Triton-100X and incubated with secondary antibodies diluted in solution 2 (10% BSA, 0.3% Triton-100X) at 37 C, for 1 hour. For IHC, additional incubation with streptavidin-b-peroxidase from horseradish (POD) conjugates at 37 C for 1 hour was performed before DAB staining. Immunofluorescence pictures were taken using Axiovert 200 (Zeiss) or Cell Observer Spinning Disk Confocal Microscope (Zeiss). IHC pictures were selected after scanning performed on Pannoramic P250 Flash III (3DHistech). Colocalization analyses and Pearson's coefficient calculations were performed using Zen Software (Zeiss).
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