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13 protocols using z0334

1

Astrocyte Immunofluorescence Staining Protocol

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Astrocyte cultures were fixed in 4% PFA/4% sucrose, permeabilized with 0.1 M PBS containing 0.1% Triton X-100 for 15 min, and blocked for 1 h with 1% bovine serum albumin in PBS at room temperature. Likewise, they were incubated with primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature. Primary antibodies used for this part include anti-GFAP (1:500; Dako Z0334) and anti-Fmrp (1:200, Abcam ab17722). Alexa 488- phalloidin (Thermo Fisher Scientific A12379) and DAPI were also applied to label F-actin and nuclei respectively.
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2

Western Blot Analysis of Astrocyte Proteins

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Proteins were first extracted from primary astrocyte cultures using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF with 1% Igepal CA-630, 1% sodium deoxycholate, 1% Triton X-100 and protease inhibitor cocktail (1:100, Sigma P8340), followed by BCA protein assay (Thermo Fisher Scientific 23,225) to determine the concentration of each sample. 100 μg of solubilized proteins were boiled in Laemmli sample buffer for 5 min, subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and subsequently transferred onto nitrocellulose membranes. The blots were incubated with primary antibodies of anti-GFAP (Dako Z0334), anti-Fmrp (Abcam ab17722) and anti-GAPDH (Millipore MAB374) overnight at 4 °C, then with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Protein bands were visualized with enhanced chemiluminescence reagents (Amersham Biosciences, Buckinghamshire, UK). The intensity of all resulting bands was quantified by densitometry using ImageJ, normalized to loading bands (GAPDH) and expressed as a percentage of WT controls. Triplicates were performed for each sample.
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3

Histological Analysis of Ischemic Stroke in Mice

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Mice (n = 6 per group) were euthanised at 48 h and 5 or 8 days after the induction of pMCAO. Brains were removed after transcardial perfusion, post-fixed by immersion for 3 h at room temperature and cryopreserved in 30% sucrose in 0.1 M phosphate buffer pH 7.4. Brains were frozen and cut in a cryostat (CM1950; Leica Biosystems) and 40-μm coronal sections were collected. Sections were air-dried and blocked at 4 °C overnight using a solution of 0.25% Triton X-100 and 5% donkey serum in Tris-buffered saline (TBS) followed by incubation with anti-glial fibrillary acidic protein (GFAP; Dako Z0334), anti-collagen IV (Abcam AB6586) and anti-neuronal nitric oxide synthase (nNOS; Thermo 61-7000) for 24 h at 4 °C. The signal was revealed using the avidin-biotin peroxidase complex (ABC) technique. A 1:150 dilution of biotinylated Lycopersicon esculentum (tomato) lectin (Sigma-Aldrich L0651) was used to stain some sections to study vessel distribution, and processed with an ABC kit followed by diaminobenzidine-nickel. The pictures were taken using a digital camera (Polaroid DMC IE, Cambridge, MA, USA) coupled to a microscope (Axioplan 2, Zeiss, Jena, Germany).
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4

Immunolabeling of Neurogenic Markers in Brain

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For the BrdU immuno-labeling, the brain sections were rinsed three times in 0.1 M PBS and incubated in 2 N HCl at 37°C for 15 min, followed by 0.1 M borate buffer (pH = 8.6) for 10 min and washed 3× with 0.1 M PBS. Sections were incubated in blocking solution for 40 min. Then, sections were incubated with some combinations of the following primary antibodies: rat anti-BrdU (1:500; AbD Serotec Cat # OBT0030, RRID:AB_609568), mouse anti-NeuN (1:500; Millipore Cat # MAB377, RRID:AB_2298772), rabbit anti-GFAP (1:100; Dako Cat # Z0334, RRID:AB_10013382), rabbit anti-Sox2 (1:500, Abcam Cat # AB97959; RRID:AB_2341193), guinea pig anti-doublecortin (DCX, 1:1000: Millipore Cat# AB2253, RRID:AB_1586992) in blocking solution at 4°C overnight. Sections were rinsed 3× with 0.1 M PBS, and incubated in 0.1 M PBS containing 10% fetal bovine serum and conjugated secondary antibodies (Alexa Fluor® 488 anti-rat Cat # A-21208; Alexa Fluor® 594 anti-rat Cat # A-11007; Alexa Fluor® 488 anti-mouse Cat # A32723; Alexa Fluor® 594 anti-rabbit; Alexa Fluor® 594 Cat# R37117; anti-guinea pig Cat# A-11076; dilution 1:1000; Thermo Fisher) for 1 h at room temperature and washed 3× with 0.1 M PBS. Nuclear counterstaining was done with 4′,6-diamidino-2-phenylindole (DAPI; Abcam Cat # ab104139, Cambridge, MA, USA).
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5

Immunofluorescence Staining of Mouse Brain

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For immunofluorescence, mice were anesthetized (ketamine 100 mg/kg and xylazine 10 mg/kg, intraperitoneally) and then intracardially perfused with 4% paraformaldehyde (PFA) diluted in 0.1 M phosphate buffer solution. The brains were removed and post-fixed in 4% PFA overnight at 4 °C. Thereafter, the brains were changed to PFA + 15% sucrose. Finally, brains were frozen in powdered dry ice and stored at − 80 °C until sectioning. Coronal brain sections of 30 μm were obtained (Leica Microsystems CM 3050S cryostat, Wetzlar, Germany) and stored in cryoprotective solution at − 20 °C until used.
Free-floating brain slices were washed 5 min in PBS and blocked and permeabilized in PBS, BSA 1% and 0,3% Triton X-100 solution for 20 min. After two washes of 5 min with PBS (0.1 M), primary antibody (GFAP (Dako/Z0334) or Iba-1 (Abcam/ab48050), were incubated over-night at 4ºC at a dilution of 1:400. The following day, after two washes with PBS, the secondary antibody (Alexa Fluor 594, Abcam/ab150080) was incubated at room temperature for 1 h in the dark at a dilution of 1:400. Later, sections were co-incubated with 1 mg/mL Hoechst (Sigma) staining solution for 5 min in the dark at room temperature and washed twice for 5 min in PBS. Finally, the slices were mounted with Fluoromount G (EMS, USA).
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6

Retinal Immunofluorescence Staining Protocol

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Immunofluorescence staining of both cryosections and whole mount retina was performed as previously reported [7 (link)]. Primary antibodies used on cryosections included anti-alpha DG (1:200, Millipore, Cat. 05-593), anti-beta DG (1:100, Abcam, ab49515), anti-LAMA1 (1:200, Millipore, MAB1903), anti-GFAP (1:500, DAKO Z0334), and anti-CD68 (1:100, Abcam, ab31630). Primary antibodies used for whole mount staining included anti-GFAP (1:100), and anti-G. simplicifolia isolectin (1:200, Invitrogen).
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7

Cryo-Tissue Analysis of Kainic Acid Injury

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Brains of the KA group, including two controls, were taken out, frozen in isopentane (− 50 °C) and cryo-cut in 20 μm slices coronally. Slices were fixed with paraformaldehyde and subsequently stained for glial fibrillary acid protein (anti-GFAP, DAKO Z0334), microglia (anti-Iba1, abcam ab107159), and neurons (anti-NeuN, Millipore MAB377) using standard protocols for fixed cryoslices. Stained slices of KA-treated rats and healthy controls were evaluated for differences in the visually striking regions determined in PET and MRI.
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8

Immunocytochemistry Protocol for Oligodendrocyte Lineage

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Cells were fixed with 4% paraformaldehyde for 20 min before permeabilization for 10 min with 0.5% Triton X-100 (Sigma #X100) in blocking media (Earl’s Balanced Salt Solution [Gibco] with 5% calf serum and 1% BSA Fraction V). Permeabilized cells were then blocked for 1 h at 25°C before overnight incubation with primary antibodies (A2B5 hybridoma [IgM, 1:4], GalC hybridoma [IgG3, 1:10], Olig2 [1:500; Millipore #MABN50], Ki67 [1:1000, BD Pharmingen #550609], GFAP [1:2000; DAKO #Z0334], Tuj1 [1:2000; Abcam #14545]) diluted in blocking media at 4°C. After washing, cells were incubated with species- and isotype-matched Alexa Fluor-conjugated secondary antibodies (1:2000; Invitrogen) and counterstained with DAPI (1 μg/mL; Invitrogen #D1306) for 30 min at 25°C before final washes with PBS and ddH2O.
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9

Cerebral Organoid Tissue Processing and Immunostaining

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Cerebral organoids were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C, then washed in PBS for 10 min and dehydrated by incubations in Ethanol (70%, 95% then 100%) for 1 h at 4 °C followed by two times 1 h incubation with Xylene 100% at room temperature. The cerebral organoids were then embedded in Paraplast Plus (Leica, 39602004) and sectioned at 30 µm. Tissue sections were stained with Haematoxylin and Eosin (H&E) or used for immunostaining. For immunofluorescence, antigen retrieval was performed by using the Antigen Retriever buffer (Citrate Buffer pH 6.0, Sigma, C 9999, 10× ). Sections were then blocked and permeabilized in blocking buffer (0.5% Triton X-100 and 1% BSA in PBS) for 20 min. Sections were then incubated with primary antibodies in blocking buffer at the following dilutions: SOX2 (mouse, R&D systems, MAB2018, 1:200), TUJ1 (mouse, Biolegend MMS-435P, 1:3000), GFAP (Rabbit, Dako Z0334, 1/2500), PCNA (Rabbit, abcam ab18197, 1 µg mL−1). For visualization, an antibody anti-mouse immunoglobulin G (anti–mouse IgG) Alexa Fluor 594 conjugate (Invitrogen, Molecular Probes) and an anti-rabbit IgG Alexa Fluor 488 conjugate (Invitrogen, Molecular Probes) was applied. DNA was stained by DAPI (1/500) and sections were mounted in ProLong Diamond Antifade mountant (Thermo Fisher Scientific, P36965). Images were collected by using an Olympus FV3000 RS with a 20x objective.
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10

Immunocytochemistry of Neuronal and Glial Cultures

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Primary neuronal, astrocytes or astrocytic-neuronal co-cultures plated on glass coverslips were washed twice with TBS and fixed at room temperature for 10 min with 100% cold methanol. Cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min and then incubated for 1 h in 5% goat serum to reduce nonspecific background. After an overnight incubation at 4 °C with the primary antibodies: [chicken or mouse anti-glial fibrillary acidic protein (Abcam, ab4674, 1: 200 or Dako, z0334, 1:500), anti-β-III-tubulin, (Abcam, ab18207, 1:500 or Covance, MMS-435P 1:1000), anti-synaptophysin (Abcam, ab106618, 1:500), anti-NeuN (Millipore, MAB377, 1:500)], cells were washed with TBS and incubated with secondary AlexaFluor-conjugated antibodies appropriate for the species (Molecular Probes, 1:500). To visualize cell nuclei, cultures were rinsed and then incubated in 4′,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI)/antifade (Sigma, 1:1000) diluted in TBS or Hoescht dye (Sigma, 1:5000) for 10 min at room temperature. Cover slips were mounted in FluorSave™ (EMD Millipore) and pictures were taken with a wide-field fluorescence microscope (Leica DMI 4000B microscope using a Leica DFC3000 G camera and the Leica application suite 4.0.0.11706).
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