SD rats were anesthetized, and the ipsilateral spinal cord was removed on day 7 after surgery. Sample sections were incubated overnight with primary antibody against NeuN-Cy3 (1:100, ABN78C3; Millipore, Temecula, CA, USA), Iba1 (1:100, ab15690; Abcam, Cambridge, UK), GFAP (1:100; Sigma-Aldrich, St. Louis, MO, USA), p62 (1:100, ab56416; Abcam), LC3B (1:100, L7543; Sigma-Aldrich), LAMP2 (1:50, PA1-655; Thermo Fisher Scientific, Waltham, MA, USA), and cleaved caspase3 (1:100, #9661S; Cell Signaling). After washing, the sections were incubated in the secondary antibody for 2 h at room temperature. Each sample section was stained with DAPI, followed by coverslips, and mounted onto slides. All samples were mounted with Fluoroshield™ with DAPI (F6057; Sigma-Aldrich; blue-fluorescent dye) and analyzed on a Zeiss LSM700 (Carl Zeiss, Oberkochen, Germany) confocal microscope with Zen software. Images were processed and quantified using Photoshop (CS6; Adobe) and ImageJ (version 1.53j; National Institutes of Health, Bethesda, MD, USA).
Abn78c3
Manufactured by Merck Group
Sourced in United States
The ABN78C3 is a laboratory instrument designed for specific functions. It is a precision device intended for use in controlled laboratory environments. No further details on its core function or intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
A11034, by Thermo Fisher Scientific (2 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (2 mentions)
Mab377c3, by Merck Group (2 mentions)
A10684, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab15690, by Abcam (1 mentions)
L7543, by Merck Group (1 mentions)
Lsm 700, by Zeiss (1 mentions)
Pa1 655, by Thermo Fisher Scientific (1 mentions)
Ab56416, by Abcam (1 mentions)
Fluoroshield with dapi, by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
Vector unmasking solution, by Vector Laboratories (1 mentions)
Ab9050, by Abcam (1 mentions)
Tunel assay, by Thermo Fisher Scientific (1 mentions)
Vs120 slide scanner microscope, by Olympus (1 mentions)
Cy3 conjugated goat anti rabbit immunoglobulin g igg, by Jackson ImmunoResearch (1 mentions)
Ab60704, by Abcam (1 mentions)
4 6 diamino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Ab178847, by Abcam (1 mentions)
7 protocols using abn78c3
1
Immunohistochemical Analysis of Spinal Cord
2
Immunostaining of Brain Sections
3
Multimodal Immunostaining of Organotypic Hippocampal Cultures
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The OHCs were immune-stained according to Gogolla and collaborators [22 (link)] using 0.05% Tween in PBS as permeabilization solution. Mature neurons were labeled with rabbit or mouse antibodies conjugated to the Cy3 fluorophore (Merck, #ABN78C3 or #MAB377C3, 1:100). Activated microglia and trimethylation of different lysine residues of histone H3 were indirectly labeled with primary antibodies anti-Iba1 (Abcam, #AB178847, 1:100), anti-H3K4me3 (Merck, #05-1339, 1:500), anti-H3K9me3 (Merck, #07-442, 1:500) and anti-H3K36me3 (Abcam, #AB9050, 1:200), respectively. As secondary antibodies, anti-rabbit (Thermo Fisher, #A11034, 1:400) or mouse (Thermo Fisher #A10684, 1:20,000) IgG conjugated with Alexa Fluor 488 fluorophore were used.
DNA fragmentation in apoptotic cells was assessed with the TUNEL assay (Thermo Fisher), performed according to the manufacturer’s instructions.
For BrdU detection, cultures were incubated in HCl 1 M for 30 min after permeabilization and stained with mouse anti-BrdU antibody conjugated to Alexa Fluor 488 (Merck, #FCMAB101A4, 1:100).
The OHCs were counterstained with DAPI (Thermo Fisher) and mounted onto glass microscope slides with ProLong Diamond Antifade Mountant (Thermo Fisher).
DNA fragmentation in apoptotic cells was assessed with the TUNEL assay (Thermo Fisher), performed according to the manufacturer’s instructions.
For BrdU detection, cultures were incubated in HCl 1 M for 30 min after permeabilization and stained with mouse anti-BrdU antibody conjugated to Alexa Fluor 488 (Merck, #FCMAB101A4, 1:100).
The OHCs were counterstained with DAPI (Thermo Fisher) and mounted onto glass microscope slides with ProLong Diamond Antifade Mountant (Thermo Fisher).
4
NeuN Antibody Staining in Gibbon Brain
5
In Vivo Immunostaining of Neurons and Microglia
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For staining neurons in vivo, the target brain slices were sealed with 10% sheep serum for 1.5 h, and immunostained with a Cy3-conjugated rabbit antibody against NeuN (diluted by PBS at 1:300, Merck Millipore, ABN78C3) overnight at 4 °C. For staining microglia cells in vivo, brain slices were blocked with 10% sheep serum in PBS with 0.3% Triton X-100 for 1.5 h, then incubated with primary antibody (anti-Iba1: diluted by PBS at 1:1000, LAK4357, WAKO) overnight at 4 °C. After washed 3 times with PBS, slices were incubated with the secondary antibody cy3-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:400, 94,600, Jackson) for 1 h at 37 °C. All the brain slices above were washed 3 times with PBS, then attached to the microscope slides and sealed with 70% glycerol. Imaging was performed by using the Olympus VS120 Slide Scanner microscope (Olympus).
6
Immunostaining Protocol for OHCs
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The OHCs were immunostained following the method described by Gogolla et al. [14 (link)] with adaptations. At the end of each experiment, the OHCs were processed for immunostaining incubating them as follows: ice-cold 4% paraformaldehyde (PFA) (pH 7.2) for 5 min, ice-cold 20% methanol in phosphate-buffered saline (PBS) 1× for 5 min, 0.05% Tween in PBS 1× overnight, and bovine serum albumin (BSA) 20% in PBS 1× overnight. Washes with 1× PBS were performed between incubations. The OHCs were removed from the inserts and incubated overnight with Cy3 conjugate anti-NeuN antibody (1:100, ABN78C3, Merck), to determine the density of mature neurons or anti-NeuroD1 (1:500, AB60704, Abcam) for post-mitotic immature neurons. To the latter, additional incubation (4 h) with a secondary antibody, Alexa Fluor 488 fluorophore conjugate anti-mouse IgG (1:20,000, A10684, ThermoFisher), was performed. Finally, the OHCs were labeled with 4′,6′-diamino-2-phenylindole (DAPI) (1 μg/mL, ThermoFisher) and slides were mounted with ProLong Diamond Antifade Mountant (ThermoFisher).
7
Quantifying Zika Virus Impact on Organotypic Hippocampal Cultures
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OHC were immune stained as previously described [27 (link)]. The density of mature neurons in OHC 8 h, 24 h, 48 h, 5, 7 and 10 days post infection (p.i.) was evaluated using Cy3-conjugated anti-NeuN antibody (1:100, MAB377C3, Merck or 1:100, ABN78C3, Merck). The presence of ZIKV NS1 protein in the OHC was determined 8, 24 and 48 h p.i. with the unconjugated anti-NS1 antibody (1:1000), kindly provided by Dr. Ada Alves from Oswaldo Cruz Institute, Fiocruz. Activated microglia morphology and density in the OHC were estimated 8 h p.i. with the unconjugated anti-Iba1 antibody (1:100, AB178847, ABCAM, Cambridge, UK). Also, the histone H3 trimethylated in the lysine 4 (H3K4me3) was quantified 5, 7 and 10 days p.i. with the unconjugated anti-H3K4me3 antibody (1:500, 05-1339, Merck). Unconjugated antibodies were subsequently labeled with anti-rabbit (1:400, A11034, ThermoFisher, Waltham, MA) or anti-mouse Alexa Fluor 488-conjugated anti-IgG antibody (1:20,000, A10684, ThermoFisher). All OHC were counterstained with 4′,6′-diamino-2-phenyl-indole (DAPI) (1 µg/mL, D1306, ThermoFisher) and slides were mounted with ProLong Diamond Antifade (P36961, ThermoFisher).
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