For IF analysis, antigen retrieval in citrate buffer (pH 6) was performed on sections, obtained as previously described, after deparaffinization and hydration. Sections were permeabilized with PBS-0.01% Triton X for 5 min at room temperature, blocked in 3% BSA for 30 min at room temperature and then incubated with αSMA primary antibody (A2547, Sigma-Aldrich) (1:250) in 0.3% BSA overnight at 4 °C.Primary antibody binding was detected incubating section with Alexa Fluor 568-labeled goat anti-mouse secondary antibody (Thermo Fisher Scientific) (1:500) for 1 h at room temperature. Hoechst was used as counterstain.
Sections were mounted in PBS-glycerol (1:1) and images were taken with Leica DMI6000B microscope. MicroVascular Density (MVD) analysis of whole hilum sections was performed with ImageJ software (NIH).
Alexa fluor 568 labeled goat anti mouse secondary antibody
Manufactured by Thermo Fisher Scientific
Alexa Fluor 568-labeled goat anti-mouse secondary antibody is a fluorescently labeled secondary antibody. It is designed to detect and visualize mouse primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Inverted fluorescence microscope, by Olympus (2 mentions)
αsma primary antibody, by Merck Group (1 mentions)
Dmi6000b microscope, by Leica camera (1 mentions)
Lsm 510 confocal microscopy, by Zeiss (1 mentions)
Rabbit anti samd9, by Merck Group (1 mentions)
Alexa fluor 488 labeled goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti ha, by Merck Group (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Dhcr7, by Abcam (1 mentions)
Lamin a, by Santa Cruz Biotechnology (1 mentions)
Phosphorylated nrf2, by Abcam (1 mentions)
Shmt1, by Abcam (1 mentions)
Pnpla3, by Abcam (1 mentions)
Mthfd2, by Proteintech (1 mentions)
Gapdh, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
5 protocols using alexa fluor 568 labeled goat anti mouse secondary antibody
1
Immunofluorescence Analysis of αSMA
2
Immunofluorescence Localization of HA and SAMD9
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N/TERT-1 and N/TERT-1 cells expressing MSCV-N-11E6 were grown on Lab-Tek Chamber slide for a day. Cells were fixed using 4% PFA and permeabilized using 0.5% Triton X-100. After blocking, cells were incubated with diluted mouse anti-HA (sigma) and rabbit anti-SAMD9 (sigma) at 4°C overnight. After three washes, cells were incubated with Alexa Fluor 568 labeled goat anti-mouse secondary antibody (ThermoFisher Scientific) and Alexa Fluor 488 labeled goat anti-rabbit secondary antibody (ThermoFisher Scientific) for 1 hr in room temperature. Prolong gold antifade mountant with DAPI (ThermoFisher Scientific) was used for mounting. Confocal images were acquired using a Zeiss LSM510 confocal microscopy with a 63x water lens. Excitation wavelengths were 364nm for DAPI, 488nm for Alexa Fluor 488, 543nm for Alexa Fluor 568 respectively.
3
Visualization of Viral Protein Expression
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MARC-145 cells were transfected with wt or mutant plasmids, and intracellular expression of viral N proteins were visualized by immunofluorescence staining at different time points post-transfection, respectively, according to a protocol described previously [27 (link)]. Cells were fixed with cold methanol, followed by blocking with 1% bovine serum albumin and incubation for 2 h with a monoclonal antibody (SR30A; Rural Technologies, INC) specifically recognizing viral nucleocapsid (N) protein. After washing with PBS, the cells were incubated for 1 h with an Alexa Fluor 568-labeled goat anti-mouse secondary antibody (Invitrogen). After a final PBS wash step, cells were visually analyzed using an Olympus inverted fluorescence microscope (Olympus, Tokyo, Japan).
4
Comprehensive Antibody Panel for HCV Pathways
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Primary antibodies were for HCV proteins: core (Institute of immunology), NS5A (Antiprot), NS3 (Antiprot), NS5A (Virogen), and NS5B (Antiprot), beta-Actin (Abcam), ACYL (Cell signaling), HMGCR (Atlas antibodies), DHCR7 (Abcam), ACACA (Santa Cruz), ELOVL5 (Novusbio), GPAM (Abcam), MTTP (Abcam), PNPLA3 (Abcam), GCK (Santa Cruz), GAPDH (Abcam), G6PD (Santa Cruz), PPAT (Proteintech), MTHFD2 (Proteintech), PSAT1 (Santa Cruz), SHMT1 (Abcam), G6PC3 (Santa Cruz), DLAT (Santa Cruz), IDH3G (Santa Cruz), ASNS (Santa Cruz), ME1 (Abcam), PCK2 (Abcam), GPT2 (Santa Cruz), NQO1 (Proteintech), GCLC (Abnova), Nrf2 (Santa Cruz), phosphorylated-Nrf2 (Abcam), Maf-G (Abcam), Lamin A (Santa Cruz). HRP-labeled secondary antibodies were used against mouse IgG, rabbit IgG, and goat IgG (Amersham) dependent on the primary antibodies for immunoblot. Alexa-fluor-568-labeled goat anti-mouse secondary antibody (Invitrogen) was used for immunofluorescence.
5
PRRSV N Protein Detection by IFA
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Indirect immunofluorescence assays (IFA) were performed as described previously (Tian et al., 2011 (link)) for the detection of nucleocapsid (N) protein in PRRSV vJX143 infected MARC-145 cells or PAMs pre-transfected with miR-26 family or mutant mimics. Cells were fixed with cold methanol followed by blocking with 1% bovine serum albumin (BSA) and then incubated for 2 h with a monoclonal antibody (SR30A, Rural Technologies) that specifically recognizes type 2 PRRSV N proteins. After washing with phosphate-buffered saline (PBS), the cells were incubated for 1 h with Alexa Fluor 568-labeled goat anti-mouse secondary antibody (Invitrogen). Cell nuclei were counterstained with 1 μg/ml of 4′, 6′-diamidino-2-phenylindole (DAPI) for 5 min. After a final PBS wash step, cells were visually analyzed using an Olympus inverted fluorescence microscope.
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