Paraffin embedded tissues were sectioned (10 µm section thickness) and mounted on superfrost slides. After deparaffinization and antigen retrieval in citrate buffer pH 6.0, immunofluorescence staining on sections was performed using rabbit polyclonal GFP antibody (#2555, 1:1000 dilution; Cell Signalling Technologies) with Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (#A-11034, 1:200 dilution; Thermo Fisher), to unambiguously detect eGFP transgene expression and differentiate from tissue background fluorescence.8 (link),14 (link) The slides were mounted using a mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (Fluoro-Gel II, #17985; Electron Microscopy Sciences), and the slides were dried and sealed. Image acquisition was performed using a Zeiss fluorescence microscope under identical conditions (20× objective; laser intensity 30%; DAPI exposure time, 50 ms; GFP exposure time, 300 ms). Lumbar spinal cord sections were additionally co-stained for neuronal nuclei protein NeuN using mouse monoclonal NeuN antibody (#MAB377, 1:1000 dilution; Millipore) with Alexa Fluor 568–conjugated goat anti-mouse secondary antibody (#A-11031, 1:200 dilution; Thermo Fisher). The slides were then scanned using NanoZoomer S60 digital slide scanner (Hamamatsu).
Alexa fluor 568 conjugated goat anti mouse secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 568-conjugated goat anti-mouse secondary antibody is a fluorescently labeled antibody that binds to mouse primary antibodies. It can be used in various immunodetection techniques, such as immunofluorescence microscopy, to visualize and localize target antigens in biological samples.
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Lab products found in correlation
Alexa fluor 488 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
Zen software, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
Nanozoomer s60 digital slide scanner, by Hamamatsu Photonics (1 mentions)
Fluoro gel 2, by Electron Microscopy Sciences (1 mentions)
Cryostat, by Leica (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Eclipse e600, by Nikon (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
Vectashield mounting media with dapi, by Vector Laboratories (1 mentions)
Anti proliferating cell nuclear antigen, by Cell Signaling Technology (1 mentions)
Anti actin antibody, by Cell Signaling Technology (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Fluorescent microscope, by Leica (1 mentions)
Alexa fluor 594 conjugated wga, by Thermo Fisher Scientific (1 mentions)
Fluoresbrite yg microspheres, by Polysciences (1 mentions)
15 protocols using alexa fluor 568 conjugated goat anti mouse secondary antibody
1
Immunofluorescence Imaging of eGFP Transgene
2
Calbindin Immunohistochemistry in Cerebellar Degeneration
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For cerebellar degeneration analysis, 30 μm sagittal sections were collected using a Leica Cryostat. To perform immunohistochemistry, sections were first prewetted with 1× PBS for 15 min. Sections were then postfixed with 4% PFA for 15 min, permeabilized with 0.25% (w/v) Triton X-100 in 1× PBS for 15 min, blocked with 10% normal goat serum in PBST for 2 h at room temperature, and incubated overnight at 4°C with mouse anti-calbindin antibody diluted in PBST containing 2% normal goat serum (1:1000 working dilution; Swant 300, Swant; RRID:AB_10000347 ). After washing the sections extensively with PBST, they were incubated with Alexa Fluor 568-conjugated goat anti-mouse secondary antibody diluted in PBST containing 2% normal goat serum (1:800 working dilution; catalog #A-11004, Thermo Fisher Scientific; RRID:AB_2534072 ) for 2 h at room temperature. The sections were then washed three times with PBST and mounted using Fluoromount-G (SouthernBiotech).
3
Immunofluorescence Staining of PRMT5 and p-Akt
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A549 and H1299 cells were cultured on glass cover slips and incubated overnight to establish adherence. The cells were fixed with 4% paraformaldehyde for 20 minutes at 37°C, followed by permeabilization in ice‐cold methanol for 10 minutes at −20°C. The cells were incubated in blocking buffer (PBS containing 5% normal goat serum and 0.3% Triton X‐100) for 1 hour at room temperature, followed by incubation with anti‐PRMT5 and anti‐p‐ser473‐Akt antibody (diluted 1:100 in blocking buffer) at 4°C overnight. Cells were washed twice for 5 minutes in PBS, and then incubated for 2 hours with Alexa Fluor 488‐conjugated goat anti‐rabbit secondary antibody (cat no. A‐11034; Thermo Fisher) for p‐ser473‐Akt and Alexa Fluor 568‐conjugated goat anti‐mouse secondary antibody (cat no. A‐11004; Thermo Fisher) for PRMT5 (diluted 1:500 in blocking buffer) at room temperature. Nuclei were stained with DAPI (cat no. D9542; Sigma) for 10 minutes at room temperature before observation. The images were captured with Nikon Eclipse E600 fluorescence microscope.
4
Collagen Quantification in Toxin-Treated Fibroblasts
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IMR90 human fibroblasts were cultured in Eagle’s Minimum Essential Medium (EMEM) (ATCC, USA) supplemented with 10% fetal bovine serum at 37 °C with 5% CO2. Cells were seeded on glass coverslips in 24-well plates for 3 days, followed by incubation with 0.5 pM of TcdA and TcdB. After 12 h or 15 h, cells were fixed in PBS with 4% paraformaldehyde for 20 min at room temperature and blocked in 10% normal goat serum (Sigma). Collagen was detected using a mix of antibodies against collagen types I, III, and V (Santa Cruz) in a 1:1:1 ratio, and Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (ThermoFisher). Glass coverslips were mounted using VECTASHIELD mounting media with DAPI (Vector Laboratories). Confocal imaging was performed on Zeiss LSM 880 confocal microscope using a 20× or 40× Plan-Apochromat objective lens (numerical aperture of 1.4) and operated with ZEN software (Carl Zeiss, Inc). For image quantification, maximum fluorescent intensity was quantified from five fields of view per coverslip at 20× magnification using ImageJ software.
5
Immunofluorescence Localization of Cellular Proteins
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A549 were cultured on glass coverslips and incubated overnight to establish
adherence. The cells were fixed with 3.7%-4% paraformaldehyde for 15 min at room
temperature and then permeabilized with ice-cold methanol for 15 min at −20 °C.
The cells were incubated in blocking buffer (PBS containing 5% normal goat serum
and 0.3% Triton X-100) for 1 h at room temperature, followed by incubation with
anti-PRMT5 (cat# sc-376937; Santa Cruz Biotechnology), anti-proliferating cell
nuclear antigen (cat# 13110; Cell Signaling Technology), anti-phospho-Ser473-Akt
(cat# 4060; Cell Signaling Technology), and anti-actin antibody (cat# 4970; Cell
Signaling Technology or cat# sc-47778; Santa Cruz Biotechnology) (diluted 1:100
in blocking buffer) at 4 °C overnight. Cells were washed 4 times for 5 min in
PBS and then incubated for 2 h with Alexa Fluor 488-conjugated goat anti-rabbit
secondary antibody (cat no. A-11034; Thermo Fisher) for p-ser473-Akt, PCNA, and
actin; Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (cat no.
A-11004; Thermo Fisher) for PRMT5 (diluted 1:500 in blocking buffer) at room
temperature. Nuclei were stained with DAPI (cat no. D9542; Sigma) for 15 min at
room temperature before observation. The images were captured with a confocal
microscopy system (LSM700, Zeiss).
adherence. The cells were fixed with 3.7%-4% paraformaldehyde for 15 min at room
temperature and then permeabilized with ice-cold methanol for 15 min at −20 °C.
The cells were incubated in blocking buffer (PBS containing 5% normal goat serum
and 0.3% Triton X-100) for 1 h at room temperature, followed by incubation with
anti-PRMT5 (cat# sc-376937; Santa Cruz Biotechnology), anti-proliferating cell
nuclear antigen (cat# 13110; Cell Signaling Technology), anti-phospho-Ser473-Akt
(cat# 4060; Cell Signaling Technology), and anti-actin antibody (cat# 4970; Cell
Signaling Technology or cat# sc-47778; Santa Cruz Biotechnology) (diluted 1:100
in blocking buffer) at 4 °C overnight. Cells were washed 4 times for 5 min in
PBS and then incubated for 2 h with Alexa Fluor 488-conjugated goat anti-rabbit
secondary antibody (cat no. A-11034; Thermo Fisher) for p-ser473-Akt, PCNA, and
actin; Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (cat no.
A-11004; Thermo Fisher) for PRMT5 (diluted 1:500 in blocking buffer) at room
temperature. Nuclei were stained with DAPI (cat no. D9542; Sigma) for 15 min at
room temperature before observation. The images were captured with a confocal
microscopy system (LSM700, Zeiss).
6
Cardiac Tissue and Cardiomyocyte Imaging
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For WGA staining, deparaffinized heart sections were rehydrated and incubated in blocking buffer (5% normal goat serum, 1% BSA in PBS) for 1 h. Cell membrane was stained with Alexa Fluor 594-conjugated WGA (ThermoFisher, #W11262, 10 μg/mL) for 1 h and washed three times in PBS before imaging.
For α-actinin staining in cell culture, NRVMs were washed twice in PBS and then fixed in 4% PFA at 4 °C for 30 min. Cells were washed another three times in PBS and permeabilized in 0.1% Triton X-100 on ice for 5 min. After three washes in PBS, NRVMs were incubated in blocking buffer (1.5% normal goat serum, 1% BSA in PBS) for 1 h and then in primary anti-α-actinin antibody (Abcam, #ab7732) for another hour. The cells were washed in PBS for three times and incubated with Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (ThermoFisher, #A-11031). Excess antibody was removed by three washes of PBS. All slides were then mounted with ProLong Gold antifade mountant with DAPI (ThermoFisher, #P36935) and imaged with a fluorescent microscope (Leica). Cross-sectional area (cardiac tissues) and cell surface area (NRVMs) were analyzed using the Image J 1.52P software.
For α-actinin staining in cell culture, NRVMs were washed twice in PBS and then fixed in 4% PFA at 4 °C for 30 min. Cells were washed another three times in PBS and permeabilized in 0.1% Triton X-100 on ice for 5 min. After three washes in PBS, NRVMs were incubated in blocking buffer (1.5% normal goat serum, 1% BSA in PBS) for 1 h and then in primary anti-α-actinin antibody (Abcam, #ab7732) for another hour. The cells were washed in PBS for three times and incubated with Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (ThermoFisher, #A-11031). Excess antibody was removed by three washes of PBS. All slides were then mounted with ProLong Gold antifade mountant with DAPI (ThermoFisher, #P36935) and imaged with a fluorescent microscope (Leica). Cross-sectional area (cardiac tissues) and cell surface area (NRVMs) were analyzed using the Image J 1.52P software.
7
Phagocytosis Assay for Microglia
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The phagocytotic capacity of BMDMs and primary microglia from trem2 transgenic mice and BV2 cells with Trem2 knockdown was detected using Fluoresbrite YG Microspheres (Polysciences). Cytochalasin D (Sigma-Aldrich) was used as the negative control and added into wells 1 hour before microsphere treatment. Cells were incubated with YG beads (1:2000 dilution in medium) for 2 hours at 37°C. The medium was then removed, and cells were washed three times with 1× PBS. Cells were fixed with 4% paraformaldehyde (PFA; ProSciTech) at 4°C for 15 min and stained with 4ith stained t 4 ( with 4% pa (DAPI) (1:5000 dilution in PBS). Primary microglia were labeled with Iba1 (GeneTex) primary antibody overnight, followed by incubation in Alexa Fluor 568–conjugated goat anti-mouse secondary antibody (1:500 dilution; Invitrogen) for 2 hours at room temperature. Images were captured on a microscope (Leica Microsystems). Bead colocalization with cells was quantified by ImageJ colocalization plugin and cell count (DAPI).
8
Quantifying Muscle Capillaries with Dual Labeling
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The number of capillaries were analyzed using double labeling of anti-endothelial cell (CD31) and dystrophin protein to visualize muscle fiber. Briefly, the sections were pre-fixed with ice-cold acetone at 4 °C for 10 min, then rehydrated with PBS. After blocking with 10 % normal goat serum (Sigma-Aldrich, Missouri, USA) for 1 h, the sections were simultaneously incubated with polyclonal rabbit anti-CD31 primary antibody (Abcam, Cambridge, UK) (1:100) and monoclonal mouse anti-dystrophin primary antibody (Sigma-Aldrich, Missouri, USA) (1:100) for 1 h, and then washed with PBS. Thereafter, Alexa Fluor 568 conjugated-goat anti-mouse secondary antibody (Invitrogen, California, USA) (1:500) and Alexa Fluor 488 conjugated-goat anti-rabbit secondary antibody (Invitrogen, California, USA) (1:500) were applied simultaneously for 1 h. After washing with PBS, the sections were post-fixed with 4 % paraformaldehyde for 10 min and counterstained with DAPI (Invitrogen, California, USA) (1:10,000) for 5 min. The stained sections were mounted with fluoroshield mounting medium (Sigma-Aldrich, Missouri, USA).
9
Immunofluorescent Staining of Tissue Sections
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Tissue sections were antigen retrieved and blocked with 10% goat serum and 0.1% Triton-X 100. For Ets1, insulin and glucagon staining, slides were incubated overnight with rabbit anti-Ets1 antibody (sc-111, lot# L1708), together with mouse anti-mouse insulin monoclonal antibody (Sigma) or with mouse anti-glucagon monoclonal antibody (Sigma), followed by detection with an AlexaFluor488-conjugated goat anti-rabbit secondary antibody and AlexaFluor568-conjugated goat anti-mouse secondary antibody (Invitrogen). Sections were counterstained with DAPI. Images were captured and analyzed using the Zeiss Confocal Microscope.
10
Immunohistochemical Staining of Beta-Amyloid
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Sections were washed in Tris-buffered saline (TBS) 3 times for 10 min each. Sections were then incubated for 30 min in a solution of 0.3% Triton X-100 in TBS to enhance membrane permeability. The sections were then transferred to a blocking solution consisting of 0.3% Triton X-100% and 5% normal goat serum and 3% bovine serum albumin in TBS and incubated for 60 min. The primary antibody solution consisted of 1:1,000 purified anti-β-amyloid, 1–16 antibody (catalog# SIG-39320, Biolegend) in 0.3% Triton X-100% and 5% normal goat serum and 3% bovine serum albumin in TBS. Sections were incubated in this solution overnight and rinsed in three changes of the Triton X-100 in TBS the following day. The sections were then transferred to a secondary antibody solution and incubated at room temperature for 2 h. This solution consisted of 1:100 Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (catalog #A-11004, Invitrogen). Following a final series of washes in TBS, the sections were mounted on gelatin-coated slides and coverslipped with an anti-fade mounting medium (Vectashield; Vector Laboratories).
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