8μm thick FFPE tissue sections consecutive from those used for LCM were stained using fluorescent immunohistochemistry. Immunostaining was performed on one section containing the hippocampus and adjacent cortex per case for each staining combination. Sections were deparaffinized and rehydrated through a series of xylene and ethanol washes. Antigen retrieval was performed by treatment with 88% formic acid for 7min, followed by boiling in citrate buffer (10mM sodium citrate, 0.05% Tween-20; pH6). Sections were blocked with 10% normal goat serum, incubated overnight at 4°C with a combination of anti-Aβ antibodies 4G8 (1:4000; BioLegend; Catalog #800709) and 6E10 (1:4000; BioLegend; Catalog #803001), and with either anti-GFAP (1:1000; Dako; Z0334) to label astrocytes, anti-SMI312 (1:500; BioLegend; Catalog # 837901) to label dystrophic neurites or anti-Secernin-1 (1:50; LSBio; Catalog # LS-C162903). All primary antibodies were diluted in 4% normal goat serum in PBS. Sections were then incubated for 2 hours at room temperature with appropriate fluorescent secondary antibodies (all diluted 1:500, from Jackson ImmunoResearch). Sections were counter stained with Hoechst 33342 (Sigma) and coverslipped (PermaFluor, Thermo Fisher Scientific).
Anti smi312
Manufactured by BioLegend
Sourced in United States, United Kingdom
Anti-SMI312 is a monoclonal antibody that binds to the SMI-312 epitope on neurofilaments, which are cytoskeletal proteins found in neurons. This antibody can be used as a tool for the detection and analysis of neurofilaments in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescent secondary antibody, by Jackson ImmunoResearch (1 mentions)
Z0334, by Agilent Technologies (1 mentions)
Ls c162903, by LSBio (1 mentions)
Permafluor, by Thermo Fisher Scientific (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Anti map2, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Axiocam mrm, by Zeiss (1 mentions)
Ab233, by BioLegend (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Axio observer, by Zeiss (1 mentions)
Vibratome, by Leica (1 mentions)
Anti map2, by GeneTex (1 mentions)
Anti gfap, by Abcam (1 mentions)
Anti parvalbumin, by Abcam (1 mentions)
Anti synaptobrevin, by Synaptic Systems (1 mentions)
Anti connexin 43, by Merck Group (1 mentions)
Fluoview 1000 laser scanning confocal microscope, by Olympus (1 mentions)
8 protocols using anti smi312
1
Fluorescent Immunohistochemistry of FFPE Brain Tissue
2
Immunofluorescent Staining of Neuronal Markers
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For immunofluorescent staining, the cells were firstly permeabilized with 0.2% Triton X-100 in PBS for 10 min after which they were blocked in 3% donkey serum in PBS for 1 h at room temperature (RT). Primary antibodies (anti-MAP2 1:1000, Abcam; and anti-SMI312 1:200, Biolegend) were diluted in 3% donkey serum in PBS and incubated overnight at 4 °C. Following washes with PBS, secondary antibodies (Alexa Fluor 488 1:1000; and Alexa Fluor 546 1:1000, Life Technologies) were diluted in 1% donkey serum in PBS and incubated for 2 h at RT. Cells were gently washed with PBS and counterstained with DAPI for 10 min at RT, washed again and mounted with DAKO fluorescent mounting medium. As negative controls for all antibodies, secondary antibody-only controls were carried out.
3
Immunofluorescence Visualization of Neuronal Markers
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Cortical neurons and cultured cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and blocked with 10% normal goat serum at room temperature for 60 min. Cells were then incubated with anti-tagRFP (1:1,000, Evrogen, AB233), anti-SMI312 (1:1,000, Biolegend, 837904), and anti-HA (1:1000, MBL, TANA2) at room temperature for 60 min. Primary antibody binding was visualized using the Alexa Fluor-conjugated secondary antibody (1:1500, Thermo). Immunofluorescence images were acquired using 40× objective lenses (NA, 0.75; Zeiss) and a CCD camera (AxioCam MRm, Zeiss) mounted on an inverted microscope (Axio Observer, Zeiss).
4
Immunofluorescence Imaging of Organoid Sections
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Organoid samples were fixed in 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH: 7.4) overnight at 4 °C. After rinsing, samples were embedded 10% agarose and sectioned (100 µm) using a vibratome (Leica, Lumberton, NJ). Primary antibodies used were anti-Parvalbumin (1:200; abcam, Cambridge, MA; ab11427), anti-GAD65 (1:200; GeneTex, Irvine, CA; GTX113192), anti-SMI312 (1:500; BioLegend, San Diego, CA; 801701), anti-GFAP (1:500; abcam, Cambridge, MA; ab53554), anti-connexin 43 (1:200; Millipore, Burlington, MA; MAB3067), anti-MAP2 (1:500; GeneTex, Irvine, CA; GTX82661), anti-synaptobrevin (1:500; Synaptic Systems, Goettingen, Germany; 104-211). Samples were viewed through a ×20 oil immersion lens (N.A. 0.85) and imaged using an upright Olympus Fluoview 1000 laser scanning confocal microscope (Center Valley, PA) equipped with a motorized stage utilizing Olympus Fluoview software version 4.2. High-resolution wide-field mosaics were produced as described elsewhere78 (link).
5
Immunostaining and Microscopy Analysis of Cryosections
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Cryosections were fixed in acetone for 10 min. Immunostaining and analysis of cryosections were performed as described previously [28 (link)]. The following antibodies were used: anti-CD3 (1:150; Bio-Rad, United States, Hercules), anti-F4/80 (1:100; Bio-Rad), anti-MBP (1:250; Sigma‒Aldrich), and anti-SMI312 (1:250; Biolegend) combined with Alexa Fluor 555-labeled anti-rat IgG and Alexa Fluor 488-labeled anti-mouse IgG secondary antibodies (1:400; Invitrogen, United States, Waltham). Analysis was carried out using a Nikon Eclipse 80i microscope and ImageJ software.
6
Immunostaining of Expanded Hydrogel Samples
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Digested gels were washed twice (20 min each time) in PBS, and then incubated in a primary antibody cocktail containing anti-SMI312 (BioLegend, 837904, 1:500 dilution), anti-MAP2 (Cell Signaling Technology, #8707, 1:500 dilution), 2% v/v normal goat serum (Jackson ImmunoResearch, 005-000-121, 1:500 dilution), and 0.1% w/v Triton X-100 in 1× PBS at 4°C for 24 hr. The gels were then washed three times (20 min each time) in wash buffer (30 mM sodium chloride, 0.1% w/v Triton X-100 in 1× PBS) and incubated in a secondary antibody cocktail containing Alexa Fluor 647 AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch, 115-605-003, 1:500 dilution), Rhodamine Red-X (RRX) AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch, 111-295-003, 1:500 dilution), 0.2% v/v normal goat serum, and 0.1% w/v Triton X-100 in 1× PBS at 4°C for 24 hr. The antibody-labeled gels were washed three times (20 min each time) in wash buffer and then placed in deionized water for expansion. For nuclei staining, propidium iodine was added to deionized water at a final concentration of 1 μg/mL during expansion.
7
Neuromuscular Junction Visualization
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The harvested extensor hallucis longus muscle was clamped with a glass slide, compressed manually, and fixed in 4% PFA in 0.1 M phosphate buffer. They were stained with anti-β3-tubulin antibody (1:500; Abcam, Cambridge, UK), anti-SMI-312 (1:500; BioLegend, San Diego, CA), Alexa Fluor 594 conjugate α-bungarotoxin (α-BTX, 1:400, Life Technologies Japan, Tokyo, Japan), and observed using confocal microscopy (TiE-A1R, Nikon, Tokyo, Japan).
8
Neuronal Immunofluorescence Staining
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Neurons were fixed in 4% paraformaldehyde/4% sucrose solution for 15 min at 37 °C, permeabilized with 0.25% Triton X-100, and blocked with 0.5% fish skin gelatin at 7 and 16 DIV26 (link). The following antibodies were used: anti-SMI312 (BioLegend 837904, mouse IgG1/IgM, 0.5 μg mL−1), anti-MAP2 [Abcam ab5392, chicken polyclonal IgY (IgG), 2.5 μg mL−1], Alexa 488-labeled goat anti-mouse IgG1 (Molecular Probes A21121, 2 μg mL−1), and Alexa 568-labeled goat anti-chicken IgG (Molecular Probes A11041, 8 μg mL−1).
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