TF-1 cells were collected on histogrip (Invitrogen, Frederick, MD, USA) coated slides by cytospin (Shandon Cytospin 3, Pittsburgh, PA, USA), were fixed with 4% paraformaldehyde for 15 min at room temperature (RT) and permeabilized in 0.1% Triton X-100 in PBS for 10 min at RT, followed by blocking with 10% donkey serum (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 h at RT. Primary antibodies were applied at the manufacturer's recommended concentrations, followed by overnight incubation in a humidified chamber at 4 °C. Fluorophore-conjugated secondary antibodies were applied at the dilution of 1:500 and incubated for 2 h at RT. The cells were mounted with Prolong Gold antifade reagent with DAPI (Invitrogen, Eugene, OR, USA) for nuclear counterstaining. Images were captured at a resolution of 2048 × 2048 using a Carl Zeiss LSM 780 (Carl Zeiss, Jena, Germany) confocal microscope with a × 40 Plan objective lens. The LSM Image Browser version 4.2.0.121 (Carl Zeiss MicroImaging, Jena, Germany) was used to analyze the microscopic slides.
Carl lsm 780
Manufactured by Zeiss
Sourced in Germany
The Carl Zeiss LSM 780 is a high-performance laser scanning microscope designed for advanced imaging applications. It features a fully integrated confocal system with a range of laser excitation sources, enabling researchers to capture detailed, high-resolution images of samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab33342, by Abcam (2 mentions)
Lsm image browser version 4, by Zeiss (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Histogrip, by Thermo Fisher Scientific (1 mentions)
Ventana benchmark automated staining system, by Roche (1 mentions)
Hrp conjugated goat anti rabbit igg mab, by ZSGB-BIO (1 mentions)
Ab78316, by Abcam (1 mentions)
Cy3 labeled goat anti rabbit igg a0516, by Beyotime (1 mentions)
Ab48394, by Abcam (1 mentions)
Ab15200, by Abcam (1 mentions)
Zen 2012, by Zeiss (1 mentions)
Fb002, by Thermo Fisher Scientific (1 mentions)
Hnrnp e1 rabbit polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
G7126, by Merck Group (1 mentions)
Rhodamine conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
α sma, by Merck Group (1 mentions)
Ab150110, by Abcam (1 mentions)
Sc 377009, by Santa Cruz Biotechnology (1 mentions)
6 protocols using carl lsm 780
1
Immunofluorescence Imaging of TF-1 Cells
2
Immunohistochemical Analysis of SEMA-7A
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HMEC-1 cells and lung endothelial tissue from experimental rats were fixed using 10% formaldehyde for 2 h at 37°C and then embedded in paraffin. For HMEC-1 cells, rat anti-human SEMA-7A antibody (1:1,000, cat no. ab23578; Abcam) labeled with fluorescein isothiocyanate (green fluorescence) for 12 h at 4°C was used to analyze SEMA-7A expression prior to and following treatment with Anti-SEMA-7A (2 mg/ml), SEMA-7A (2 mg/ml) or PBS (2 mg/ml) for 2 h at 4°C. Lung tissues were cut into sections 4-µm thick. Antigen retrieval was performed in tissue sections and the sections were incubated with the following primary antibodies: SEMA-7A (1: 1,000; cat no. ab23578), M1-7 (1:1,000; cat no. ab61108) and FSF/FKF (1:1,000; cat no. ab200478; all Abcam) for 8 h at 4°C and correlative secondary antibodies: HRP-conjugated goat anti-rabbit IgG mAb (1:2,000; cat no. PV-6001; ZSGB-BIO, Beijing, China) were applied for specimens for 24 h at 4°C. The staining of the slides was performed with the avidin-biotin-peroxidase complex. A Ventana Benchmark automated staining system (Version 3.0, Ventana Medical Systems, Inc; Roche Diagnostics, Basel, Switzerland) was used to analyze SEMA-7A, ERF, ERK, M1-7 and FSF/FKF levels using confocal microscopy (Carl Zeiss LSM780, Carl Zeiss AG, Oberkochen, Germany).
3
Immunofluorescence Analysis of p53 and LC3-II
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After incubation with SePTX NPs for 48 h, the cells were incubated in 4% paraformaldehyde for 20 min, 1% of bovine serum albumin was used for cell blocking, p53 (ab78316; Abcam) or LC3-II (ab48394; Abcam) was used for incubation of primary antibodies. The blocking time was 30 min at room temperature and the primary antibody was incubated overnight at 4oC. Cy3-labeled goat anti-rabbit IgG (A0516; Beyotime) was used for incubation of the secondary antibody, and the secondary antibody were incubated for 1.5 h at 4oC. DAPI was used for staining of nuclei, and confocal microscopy (Carl Zeiss LSM780; Instrument Development Center, NCKU, Tainan City, Taiwan) was used for observation of intracellular fluorescence intensity.
4
Phenotypic Validation of Primary Human Myoblasts and Fibroblasts
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To confirm primary human myoblast (PHM) and primary human fibroblast (PHF) phenotype and culture purity, cells were fixed with 4% PFA, blocked, and stained overnight with desmin (ab15200, Abcam, UK) and fibronectin (sc80982, Santa Cruz, USA) at 4°C. Cells were then stained with fluorescence-labelled secondary antibodies (594–150064 and 488–150109, Abcam, UK) and Hoechst (ab33342 Abcam, UK), mounted with fluorescent mounting media (53023, DAKO, Denmark), and imaged on the Zeiss confocal microscope (Carl Zeiss LSM 780, Zeiss, Germany) at 200x magnification. PHM phenotype was confirmed by positive staining with desmin only, while PHF phenotype was confirmed by positive staining with fibronectin only (Figure 1 ).
5
Immunofluorescent Localization of hnRNP E1
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Cells were cultured to 50–70% confluence on glass-bottom tissue culture dishes (150680; Thermo Fisher Scientific) and then washed with phosphate-buffered solution (PBS, 10010023; Thermo Fisher Scientific). The cells were fixed with 2% paraformaldehyde (FB002; Thermo Fisher Scientific), followed by the addition of glycine buffer (0.1 mM glycine) (G7126; Sigma-Aldrich) for paraformaldehyde quenching and blocked in 1% bovine serum albumin (BSA, TS-38839; Thermo Fisher Scientific) in PBS for 1 h. The hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology) was diluted in PBS (1:200) with 1% BSA and incubated with the cells at 4°C overnight. The cells were then washed with cold PBS three times prior to incubation with rhodamine-conjugated goat anti-rabbit IgG (1:500; Pierce Biotechnology) for 45 min at room temperature. The labeled cells were washed with cold PBS and imaged using a confocal microscope (Carl Zeiss LSM780; Carl Zeiss Microscopy GmbH, Jena, Germany). The images were analyzed using ZEN 2012 software (Carl Zeiss Microscopy GmbH).
6
Immunohistochemistry of Tissue Sections
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Tissue sections were fixed in 4% paraformaldehyde (158127, Sigma‐Aldrich, USA), washed and blocked for 90 minutes in 5% donkey serum (S217G, Celtic Diagnostics, South Africa) in 1% bovine serum albumin (BSA, 10735086001, Roche, Germany), with 0.2% Triton‐X‐100 (X100, Sigma‐Aldrich, USA). Sections were incubated with primary antibodies for Pax7 (1:50, Pax7, Dev. Studies Hybridoma Bank, USA), F4/80 (1:200, sc377009, Lot#I1317, Santa Cruz, USA), and α‐smooth muscle actin (1:250, α‐SMA, Lot#125M4797V, A2547, Sigma‐Aldrich, Germany) in 1% BSA overnight at 4°C. After washing, secondary antibodies were added for 2 hours (Donkey Anti‐mouse 555, 1:500, ab150110, Abcam). Tissues were stained with Agglutinin (W11261, Thermo Fisher Scientific, USA) and Hoechst stain (ab33342, Abcam, UK).
Imaging was performed on a Confocal Microscope (Carl Zeiss LSM 780, Zeiss, Germany) and analyzed on Zen 2011 software (Zeiss, Germany). The EC Plan‐Neofluar 10x/0.3 M27 and Plan‐Apochromat 20x/0.8 M27 objectives were used to acquire images at 100x and 200x magnification, respectively. (Magnification represented as ocular lens (10x) multiplied by objective lens (10x/20x)).
Imaging was performed on a Confocal Microscope (Carl Zeiss LSM 780, Zeiss, Germany) and analyzed on Zen 2011 software (Zeiss, Germany). The EC Plan‐Neofluar 10x/0.3 M27 and Plan‐Apochromat 20x/0.8 M27 objectives were used to acquire images at 100x and 200x magnification, respectively. (Magnification represented as ocular lens (10x) multiplied by objective lens (10x/20x)).
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