Immunostaining was performed as described earlier [30 (link), 31 (link)]. Briefly, coverslips containing 200–300 cells/mm2 were fixed with 4 % paraformaldehyde for 15 min, followed by treatment with cold methanol (−20 °C) for 5 min and two rinses in PBS. Samples were blocked with 2 % BSA in PBS containing Tween 20 (PBST) for 30 min followed by incubation in PBST containing 1 % BSA and mouse anti-CD68 (1:200), goat anti-IBA1 (1:200), rabbit anti-iNOS (1:200), mouse anti-GFAP (1:500), mouse anti-MBP (1:200), and goat anti-MAP-2 (1:100). After three washes in PBST (15 min each), slides were further incubated with Cy2, Cy5, and DAPI (Jackson ImmunoResearch, West Grove, PA, USA). After secondary antibody incubation, coverslips were rinsed in 1X PBS, mounted on slides in Fluoromount (Sigma) and imaged using an Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera.
Bx41 fluorescent microscope
Manufactured by Hamamatsu Photonics
The BX41 is a fluorescent microscope manufactured by Hamamatsu Photonics. It is designed to enable visualization and analysis of fluorescently labeled samples. The microscope features LED illumination, multiple filter sets, and a high-resolution camera for capturing images and videos.
Automatically generated - may contain errors
Lab products found in correlation
Orca 03g camera, by Hamamatsu Photonics (9 mentions)
Fluoromount, by Merck Group (5 mentions)
Thio s, by Merck Group (2 mentions)
Cy2 or cy5 conjugated secondary antibodies, by Jackson ImmunoResearch (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Jackson ImmunoResearch (1 mentions)
Fluormount, by Merck Group (1 mentions)
Cy5 labeled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
9 protocols using bx41 fluorescent microscope
1
Immunostaining Protocol for Cell Visualization
2
Immunofluorescence and Thioflavin-S Staining in Mouse Brain
3
Immunofluorescence and Thioflavin-S Staining in Mouse Brain
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It was performed as described before.60 (link),63 (link) Briefly, mice were anesthetized and perfused with PBS (pH 7.4) and then with 4% (w/v) paraformaldehyde solution in PBS followed by dissection of the brain from each mouse for IHC.64 (link),65 (link) Samples were incubated in PBS containing 0.05% Tween 20 (PBST) and 10% sucrose for 3 h and then 30% sucrose overnight at 4°C. Brain was then embedded in O.C.T (Tissue Tech) at −80°C, and processed for conventional cryosectioning. Frozen sections (30 μm) were treated with cold ethanol (−20°C) followed by two rinses in PBS, blocking with 3% BSA in PBST and double labeling with two antibodies (Table S1 ). After three washes with PBST, sections were further incubated with Cy2 or Cy5 conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). The samples were mounted and observed under the Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera. During Thio-S staining, after washing and incubation with secondary antibodies for 1 h, free-floating sections were stained with 0.002% Thio-S (Sigma) made up in TBS for 8 min. Sections were washed twice in 50% EtOH for 1 min and twice in TBS for 5 min before drying and mounting in Fluoromount (Sigma).
4
Immunofluorescence Staining of Astrocytes
5
IFN-γ Immunocytochemistry in A549 Cells
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Immunocytochemistry was performed by plating coverslips containing A549 cells cultured to 70–80% confluence as described before [19 (link),20 (link),21 (link)]. The cells were fixed with chilled Methanol (Fisher Scientific, Waltham, MA, USA) for one hour, followed by rinses with filtered PBS. Samples were blocked with 2% BSA (Thermo Fisher, Waltham, MA, USA) in PBS containing Tween 20 (Millipore Sigma, Burlington, MA, USA) and Triton X-100 (Millipore Sigma, Burlington, MA, USA) for 30 min and incubated at room temperature on a shaker. The primary antibodies used included: IFN-γ (1:100; Thermo Fisher, Waltham, MA, USA) incubated for 2 h on a shaker. After multiple washes in filtered PBS, coverslips were incubated with Cy5- labeled secondary antibody (1:200; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After four washes in PBS, cells were incubated for 5 min in 4′,6′- diamindino-2- phenylindole (DAPI, 1:10,000; Millipore Sigma, Burlington, MA, USA). The coverslips were mounted and dried overnight then observed the Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera.
6
Immunofluorescence Analysis of Free-Floating Brain Sections
7
Detecting Apoptosis-Induced DNA Fragmentation
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Fragmented DNA was detected in situ by the terminal deoxynucleotide transferase (TdT)-mediated binding of 3′OH ends of DNA fragments generated in response to apoptotic signals, using a commercially available kit (TdT FragEL DNA Detection Kit) from Millipore Sigma (Burlington, MA, USA) as described earlier [16 (link),17 (link),18 (link)]. Coverslips containing A549 lung adenocarcinoma cells cultured to 70–80% confluence were fixed with chilled methanol (Fisher Scientific, Waltham, MA, USA) for an hour, followed by two brief rinses with sterile PBS. Cover slips were treated with 20 mg/mL proteinase K for 5 min at room temperature and washed in PBS before TdT staining. Samples were equilibrated for 30 min in 1xTdT buffer and washed with PBS prior to terminal deoxynucleotidyl transferase and DAPI (1:10,000, Millipore Sigma, Burlington, MA, USA) staining. After mounting coverslips and drying overnight, slides were visualized under the Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera.
8
Immunofluorescence Staining of Primary Hippocampal Neurons
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Primary hippocampal neurons were washed three times with 1X PBS, fixed in 4% paraformaldehyde for 10 min or with chilled methanol overnight, washed again with 1X PBS and incubated first with primary antibodies (Table S1 ) followed by Cy2 or Cy5 conjugated secondary antibodies. After secondary antibody incubation, coverslips were rinsed in 1X PBS, mounted on slides in Fluoromount (Sigma) and imaged using an Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera.
9
Immunofluorescence Staining of Primary Hippocampal Neurons
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