Free floating 45 μm thick sagittal sections were cut using a Leica SM2010 R sliding microtome and transferred to sterile TBS for storage. Sections were gathered and placed sequentially into wells (∼4 per well). Sections were then randomly selected from each well to perform antibody staining using the primary antibodies ACU193 (0.2 μg/ml), Alexa Fluor® 555-conjugated NU4 (0.92 μg/ml), Cy3-conjugated anti-GFAP (1:800, Sigma) and the secondary antibody Alexa Fluor® 633 goat anti-human IgG (1:2000, Invitrogen). Floating slices were rinsed 3 × 10 min with TBS and blocked with blocking buffer (10% NGS with 0.3% Triton X-100 in TBS) for 60 min at RT. Slices were then incubated with the respective antibodies in blocking buffer overnight at 4°C with gentle rotation. Sections were washed 3 × 10 min in TBS and incubated with secondary antibody for 3 h at RT with orbital agitation in the dark. Secondary was prepared in blocking buffer diluted 10-fold with TBS. Sections were then washed 3 × 10 min in TBS, mounted using ProLong Diamond® antifade mounting media with DAPI (Invitrogen) and 24 × 60 mm No.1.5 glass coverslips (Thermo Scientific). Z-stacks of the brain sections were collected at 10× or 100× on a Leica SP5 confocal microscope and analyzed with ImageJ.
Prolong diamond antifade mounting media with dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States
ProLong Diamond antifade mounting media with DAPI is a ready-to-use, long-lasting mounting solution designed for fluorescence microscopy. It contains DAPI, a nuclear stain that emits blue fluorescence when bound to DNA, allowing for visualization of cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Glial fibrillary acidic protein (gfap), by Agilent Technologies (2 mentions)
Formalin, by Merck Group (2 mentions)
Species specific biotinylated conjugated secondary antibodies, by Vector Laboratories (2 mentions)
Streptavidin hrp vectastain elite abc kit, by Vector Laboratories (2 mentions)
Fluorescence conjugated alexa fluor secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Sm2010r sliding microtome, by Leica (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Cy3 conjugated anti gfap, by Merck Group (1 mentions)
Alexa fluor 488, by Merck Group (1 mentions)
Active caspase 3, by Cell Signaling Technology (1 mentions)
Neuronal nuclei (neun), by Abcam (1 mentions)
Oil immersion objective, by Nikon (1 mentions)
Bz 800 microscope, by Keyence (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Anti synaptophysin, by Synaptic Systems (1 mentions)
Sat704a001ea, by PerkinElmer (1 mentions)
Ab62341, by Abcam (1 mentions)
Z0334, by Agilent Technologies (1 mentions)
9 protocols using prolong diamond antifade mounting media with dapi
1
Immunohistochemical Analysis of Brain Sections
2
Histological and Immunohistochemical Analysis of P7 Mouse Brains
3
Visualizing Tau Oligomer Interactions in Cortical Neurons
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Primary cortical neurons from C57BL/6 mice (Jackson Laboratory, stock#000664) were plated at a density of 1 × 106 cells/mL using poly-L-lysine coated coverslip in 24-well plates as previously described83 (link). Cortical neurons were treated for 1 hour with 0.5 µM TauO labeled with Alexa Fluor-568 or 0.5 µM TauO labeled with Alexa Fluor-568 pretreated with 5 µM of curcumin derivatives. After washing off unbound proteins, cells were fixed in 4% paraformaldehyde for 15 min at room temperature. Neurons were then washed three times with 1X PBS (10 min each) and permeabilized with 0.25% Triton-X 100, diluted in 1X PBS for 10 min. After washing three times with 1X PBS, neurons were blocked with 5% goat serum for 1 hour. Neurons were then immunostained overnight with the mature neuronal marker, β-III Tubulin (1:1000; ab78078, Abcam) at 4 °C. The next day, neurons were washed three times with1X PBS and incubated with goat anti-mouse IgG Alexa Fluor-488 (1:500, Thermo Fisher Scientific) for 1 hour at room temperature. After washing with 1X PBS, neurons were mounted using Prolong Diamond Antifade mounting media with DAPI (P36966, Invitrogen). Slides were then dried in fume hood. Cortical neurons were imaged with a Keyence BZ-800 Microscope using standard filters for DAPI, GFP and Texas Red. Nikon 100X oil immersion objective was used to capture images.
4
Immunostaining and Imaging of Mouse Spinal Cords
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Spinal cords were recovered and stained as previously described60 (link),75 (link). Following terminal anesthesia by pentobarbital, mice were perfused transcardially with 10% formalin (Sigma). Spinal cords and brains were removed, post-fixed overnight, transferred to buffered 30% sucrose for 48 h, embedded in O.C.T. Compound (Tissue-Tek, Sakura-Finetek/VWR) and cryostat-sectioned at 30 μm. Serial horizontal sections of spinal cord containing the lesion sites and brain containing the viral injection sites were cut and processed for immunostaining. The following primary antibodies were used: anti-GFAP (DAKO Z0334, 1:1000, free-floating), anti-GAP43 (1:1000, Benowitz lab), anti-Synaptophysin (Synaptic Systems 101004, 1:1000, free-floating), and RFP (1:500, Abcam ab62341, free-floating). BDA tracing was visualized with streptavidin-HRP (1:300, PerkinElmer SAT704A001EA) antibodies plus Cy3-TSA (1:200, PerkinElmer SAT704A001EA). Sections were cover-slipped using Prolong Diamond Antifade Mounting media with DAPI (ThermoFisher) to stain cell nuclei. Each section was imaged on ZEISS LSM 880.
5
Immunostaining Procedure for Animal Tissues
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We purchased pyridostigmine bromide (PB), permethrin (Per), Neomycin trisulfate hydrate, Enrofloxacin, and Ribavirin from Sigma-Aldrich (St. Louis, MO, USA). Anti-claudin-2, anti-MyD88, anti-MCP-1, and anti-β-actin primary antibodies were purchased from Abcam (Cambridge, MA, USA). anti-β-tubulin, anti-TLR3, anti-TLR7, anti-IKKα, anti-p65, and anti-IL6 primary antibodies were purchased from Santacruz Biotechnology (Dallas, TX, USA) while anti-TRAF6 was purchased from Abclonal Technology (Woburn, MA, USA). Species-specific biotinylated conjugated secondary antibodies and Streptavidin-HRP (Vectastain Elite ABC kit) were purchased from Vector Laboratories (Burlingame, CA, USA). Fluorescence-conjugated (Alexa Fluor) secondary antibodies and ProLong Diamond antifade mounting media with DAPI were purchased from Thermofisher Scientific (Grand Island, NY, USA). All other chemicals used in this study were purchased from Sigma unless otherwise specified. Animal tissues were paraffin-embedded and sectioned into slides by AML laboratories (Baltimore, MD, USA).
6
Immunofluorescence Microscopy Protocol for Adherent Cells
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For all immunofluorescence microscopy, cells were seeded in 24-well plates on glass coverslips precoated with 0.1% gelatin (HEK293T or C2C12 cells) or 1 mg/ml Matrigel for primary cells. At the end of all experimental time-points, cells were fixed with 4% paraformaldehyde (PFA, EM grade, Electron Microscopy Services) in 1× PBS for 15 min before wells were washed three times with 1× PBS. Samples were subsequently permeabilized with 0.2% Triton X-100 in 1× PBS for 10 min at room temperature before blocking in 10% normal goat serum in 1× PBS for 1 hr. Primary antibodies indicated were diluted in 1× PBS and subsequently added to all wells. Plates were incubated overnight in a humidified chamber at 4°C, before wells were washed with 1× PBS three times. Secondary antibodies (Alexa Fluor, Thermo Fisher) were then added to the wells, and incubation proceeded for 1 hr at room temperature. The wells were washed three times with 1× PBS, before coverslips were mounted with ProLong Diamond Antifade mounting media with DAPI (Thermo Fisher) onto glass slides. After coverslips were allowed to cure overnight at room temperature, the slides were imaged with confocal microscopy (Nikon A1R). Images were analyzed and quantified using calibrated Fiji software.
7
Immunohistochemical Analysis of Drosophila Tissues
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Larval fillet and adult thoraces dissections were performed in PBS and fixed in 4% formaldehyde, pH 7.0, for 20 (larval fillet) or 30 (adult flight muscles) minutes, respectively. Permeabilisation was performed for 30 min in PBS containing 0.3% (v/v) Triton X‐100 (PBS‐TX), then tissues were blocked for 1 h in PBS‐TX containing 1% (w/v) BSA (larval fillet) or 4% Horse Serum (adult flight muscles). Primary antibody incubation was performed overnight at 4°C in PBS‐TX containing 1% (w/v) BSA. The tissues were washed 3 times for 10 min each in PBS‐TX prior to incubation with secondary antibodies for 2 h at room temperature (larval fillet) or overnight at 4°C (adult thoraces). The tissues were washed three times for 10 min in PBS‐TX, then once for 10 min in PBS, and rinsed once in water prior to mounting in Prolong Diamond Antifade mounting media with DAPI (Thermo Fisher Scientific).
8
Neuro-Inflammatory Markers and Microbiome Analysis
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We purchased PB and permethrin from Sigma-Aldrich (St. Louis, MO). Anti-RAGE, anti-Claudin 5, anti-HMGB1, anti-IL-1β, and anti-ASC-2 were purchased from Santacruz Biotechnology (Dallas, TX), Anti-BDNF from cell signaling technology (Danvers, MA), while anti-NLRP3, anti-3-nitrotyrosine, anti-IL-6, anti-IL-18, anti-TMEM 119 primary antibodies were purchased from Abcam (Cambridge, MA). Species specific biotinylated conjugated secondary antibodies and Streptavidin-HRP (Vectastain Elite ABC kit) were purchased from Vector Laboratories (Burlingame, CA). Fluorescence conjugated (Alexa Fluor) secondary antibodies, ProLong Diamond antifade mounting media with DAPI and Pierce LAL chromogenic endotoxin quantitation kit were bought from Thermo Fisher Scientific (Waltham, MA) while enzyme-linked immunosorbent assay (ELISA) kits were purchased from ProteinTech (Rosemond, IL). Unless otherwise specified, all other chemicals used were purchased from Sigma. Paraffin-embedding of tissue and sectioning were done by AML laboratories (Baltimore, MD) and at the Instrument Resources Facility, University of South Carolina School of medicine (Columbia, SC). Microbiome analysis was done by Cosmos ID (Rockville, MD).
9
Spinal Cord Tissue Preparation and Staining
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Spinal cords were recovered and stained as previously described (8, 109) . Following terminal anesthesia by pentobarbital, mice were perfused transcardially with 10% formalin (Sigma). Spinal cords and brains were removed, post-fixed overnight, transferred to buffered 30% sucrose for 48 h, embedded in O.C.T. Compound (Tissue-Tek, Sakura-Finetek/VWR) and cryostat-sectioned at 30 μm. Serial horizontal sections of spinal cord containing the lesion sites and brain containing the viral injection sites were cut and processed for immunostaining. The following primary antibodies were used: GFAP (DAKO, 1:1000, free-floating), GAP43
(1:1000, Benowitz lab), Synaptophysin (Synaptic Systems, 1:1000, free-floating), RFP
(1:500, Invitrogen, free-floating), and NeuN (1:500, Millipore, free-floating). BDA tracing was visualized with streptavidin-HRP (1:300, PerkinElmer) antibodies plus Cy3-TSA
(1:200, PerkinElmer). Sections were cover-slipped using Prolong Diamond Antifade
Mounting media with DAPI (ThermoFisher) to stain cell nuclei.
(1:1000, Benowitz lab), Synaptophysin (Synaptic Systems, 1:1000, free-floating), RFP
(1:500, Invitrogen, free-floating), and NeuN (1:500, Millipore, free-floating). BDA tracing was visualized with streptavidin-HRP (1:300, PerkinElmer) antibodies plus Cy3-TSA
(1:200, PerkinElmer). Sections were cover-slipped using Prolong Diamond Antifade
Mounting media with DAPI (ThermoFisher) to stain cell nuclei.
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