Cells were fixed into 96-well glass bottom plates (6055302, Perkin Elmer) in 4% formaldehyde (10231622, Fisher Scientific) for 15 min at room temperature. Cells were washed thrice with PBS and then permeabilised in PBS with 1% BSA (A2153, Sigma-Aldrich) and 0.5% Triton X-100 (X100, Sigma-Alrich) for 1 h at room temperature. Cells were washed thrice with PBS before overnight incubation at 4°C in PBS with 1% donkey serum (ab7475, Abcam) and 1:200 Anti-EKLF/KLF1 antibody (ab2483, Abcam). Cells were then washed thrice with PBS and incubated for 1 h at room temperature in PBS with 1% donkey serum (ab7475, Abcam) and 1:1,000 Donkey anti-Goat IgG (H + L) Cross-Absorbed Secondary Antibody, Alexa Fluor 647 (A-21447, Invitrogen). Cells were washed once with PBS and incubated with 1:1,000 DAPI (D9542, Sigma-Aldrich) for 5 min at room temperature. Cells were washed twice with PBS and stored in PBS at 4°C before imaging. Cells were imaged at 40X on the Opera Phenix® Plus High-Content Screening System and processed with Fiji software.
Ab7475
Manufactured by Abcam
Sourced in United Kingdom
Ab7475 is a biotin-conjugated monoclonal antibody that can be used to detect and quantify target proteins in various applications, including Western blotting, immunohistochemistry, and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Ab150073, by Abcam (5 mentions)
Sc 281692, by Santa Cruz Biotechnology (4 mentions)
D9542, by Merck Group (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Antifade mounting medium with dapi, by Vector Laboratories (3 mentions)
Dm 5000 b ctr 5000, by Leica (3 mentions)
96 well microplate, by Corning (2 mentions)
Tcs sp8 confocal microscope, by Leica (2 mentions)
Triton x 100 x100, by Merck Group (2 mentions)
Ab104139, by Abcam (2 mentions)
Anti fus antibody pa5 52610, by Thermo Fisher Scientific (2 mentions)
Lsm 800, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Ab8898, by Abcam (2 mentions)
Ab8580, by Abcam (2 mentions)
Sc 6215, by Santa Cruz Biotechnology (2 mentions)
Ab16048, by Abcam (2 mentions)
Ab28364, by Abcam (2 mentions)
A11058, by Thermo Fisher Scientific (2 mentions)
40 protocols using ab7475
1
EKLF/KLF1 Immunofluorescence Staining
2
Multiplex Immunofluorescence Staining of Astrocytes
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Tissue sections and astrocyte cultures obtained from rat, wild type and CB1-R knock out mice spinal cords were first incubated with a mixture of antibodies that contained mouse anti-GFAP (1:1000, catalog no.: MAB3402, Millipore, Temecula, California, USA), rabbit anti-CB1-R (1:1000, catalog no.: 10006590, Cayman Chemical, Ann Arbor, Michigan, USA) and goat anti-DGLα (1:500, catalog no.: Af1080, Frontier Institute, Hokkaido, Japan). The sections and cultures were then transferred for overnight incubation in a mixture of donkey anti-mouse IgG conjugated with Alexa Fluor 488 (1:1000, catalog no.: A-21202, Invitrogen, Eugene, Oregon, USA), donkey anti-rabbit IgG conjugated with Alexa Fluor 647 (1:1000, catalog no.: A-31573, Invitrogen, Eugene, Oregon, USA) and donkey anti-goat IgG conjugated with Alexa Fluor 555 (1:1000, catalog no.: A-21432, Invitrogen, Eugene, Oregon, USA) secondary antibodies. Before the antibody incubations, the sections were kept in 10% normal donkey serum (catalog no.: ab7475, Abcam, Cambridge, UK) for 50 minutes. Antibodies were diluted in 10 mM TPBS (pH 7.4) containing 1% normal donkey serum (catalog no.: ab7475, Abcam, Cambridge, UK). Sections were mounted on glass slides and covered with VectaShield-DAPI (catalog no.: H-1200, Vector Laboratories., Burlingame, California, USA).
3
Immunocytochemistry of Neuronal Cells
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Cells were washed for 5 minutes in PBS and then fixed with 4% (w/v) paraformaldehyde (Thermo Fisher Scientific, 28908) for 15 minutes, followed by three washes, 5 minutes each, in PbTr (PBS with 0.125% Triton-X 100; Millipore Sigma, 9400). Following an additional 10 minutes permeabilization with PbTr, the cells were blocked in PbTr with 5% normal donkey serum (Abcam, ab7475) for 45 minutes. Primary antibodies (ChAT, 1:100, EMD Milipore AB144P; Tuj1, 1:1000, BioLegend 801202) were diluted in blocking buffer and cells were incubated overnight at 4 °C in a humidified chamber. After three 5 minute washes with PbTr, cells were incubated in the dark for an hour with secondary antibodies (Alexa Fluor secondary antibodies, ThermoFisher Scientific, at a 1:1000 dilution). Cells were finally washed three times with PbTr, and the slides were mounted in Prolong Diamond with DAPI (Life Technologies, P36962). Slides were mounted overnight at room temperature prior to imaging.
4
CUBIC Optical Clearing of Cortico-Striatal Assembloids
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To optically clear cortico-striatal assembloids, we applied the hydrophilic chemical cocktails-based CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocol64 (link), 65 (link). Briefly, cortico-striatal assembloids at day 83 were fixed with 4% PFA/4% sucrose/PBS at 37°C for 20 min and incubated at 4°C overnight. Next day, the assembloids were washed twice with PBS and incubated in Tissue-Clearing Reagent CUBIC-L (TCI, T3740) at 37°C for 2 days. Assembloids were washed three times with PBS for 2 hours, then stained with anti–GFP (1:1000 dilution) and anti–mCherry (1:1000 dilution) antibodies in PBS containing 0.2% Triton X-100 (Millipore Sigma, T9284–100ML) and 3% NDS (Abcam, ab7475) at 37°C for 2 days. Assembloids were subsequently washed three times with PBS for 2 hours, then incubated with secondary antibodies (Alexa, 1:1000 dilution) in PBS containing 0.2% Triton X-100 and 3% NDS at 37°C for 2 days. For refractive index (RI) matching, assembloids were incubated with Tissue-Clearing Reagent CUBIC-R+ (TCI, T3741) at room temperature for 2 days. CUBIC-cleared assembloids were then transferred into a well of Corning™ 96-Well microplate (Corning, 4580) in 150 μL of CUBIC-R+ solution and imaged using a 10x objective on a Leica TCS SP8 confocal microscope.
5
Immunofluorescent Staining of FFPE Slides
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If formalin-fixed, paraffin-embedded slides were used for immunofluorescent staining, they were treated equally as for immunohistochemistry until incubation with different primary antibodies (LCN2: #AF3508, R&D System, 1:40; STAR: #12225-1-AP, ProteinTech Germany GmbH, Planegg-Martinsried, Germany, 1:50) or negative controls (normal goat IgG control, AB-108-C, R&D Systems; rabbit IgG control, #30000-0-AP, ProteinTech) overnight at 4 °C. 5% normal donkey serum (#ab7475, Abcam, Cambridge, UK) was used in the blocking solution (compare Section 4.5.2 ). Samples were protected from light during the consecutive staining steps to prevent unnecessary loss of fluorescent signals. Fluorescence-conjugated secondary antibodies (Table S2 ), diluted at a ratio of 1:300 in PBS, were applied on tissue samples for 1 h at RT. Subsequently, counterstaining was performed with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (#D1306, Thermo Fisher Scientific). Slices were mounted in aqueous PermaFluorTM mounting medium (#TA-030-FM, Thermo Fisher Scientific). Control slides were treated equally except for incubation with H2O instead of a primary antibody. Stained slides, which gradually lose intensity after a couple of days, were stored in the dark at 4 °C for a short period of time prior to stable documentation (see Section 4.5.4 ).
6
Immunofluorescence Staining of TPCN1, TPCN2, and TACE
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DC2.4 cells plated on coverslips were fixed with 4% paraformaldehyde for 10 min, washed three times with phosphate-buffered saline (PBS) for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, blocked by 10% donkey serum (Abcam, ab7475) for 1 hour, and then incubated with anti-TPCN1 (1:100, Abcam, ab94731), anti-TPCN2 (1:100, Abcam, ab119915), or anti-TACE (1:50, Abcam, ab13535) antibody for 1 hour at room temperature, followed by incubation with donkey anti-goat immunoglobulin G (IgG) H&L (TRITC, Abcam, ab6882) or donkey anti-rabbit IgG H&L (Alexa Fluor 488, Abcam, ab150073) secondary antibodies (1:2000) for 1 hour. After washing three times with PBS, cells were finally mounted on glass slides using ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Confocal images were acquired with a laser scanning confocal microscope (Zeiss LSM).
7
Immunofluorescence Visualization of Serotonin in Brainstem
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After the behavioral tests, the animals were anesthetized with chloral hydrate (3.5 mg/kg, intraperitoneal injection) and transcardially perfused with 150 ml pre-cooled saline followed by 400 ml of a 4% paraformaldehyde solution. The brains were removed and post-fixed in paraformaldehyde for 24 h prior to being placed in a 15% sucrose solution overnight. The brains were transferred to a 30% sucrose solution and incubated for 72 h prior to embedding in optimal cutting temperature matrix. Cryostat sections were cut at 30 µm around the DRN region (bregma −7.50, 8.00 and 8.50 mm) on a sliding microtome and blocked in 5% donkey serum (ab7475; Abcam, Cambridge, UK) in Tris-buffered saline containing Tween-20 (TBST)/Triton for 60 min. The sections were then incubated at 4°C overnight in TBST containing an anti-5-HT primary antibody (1:100 dilution; cat. no. ab10385; Abcam). Immunoreactivity to the antigen was visualized using an Alexa Fluor 488-conjugated secondary antibody (1:1,000 dilution; cat. no. 103715; Jackson ImmunoResearch Laboratories, Inc., Bar Harbor, ME, USA). Images were obtained using a fluorescence microscope (Olympus IX71; Olympus, Tokyo, Japan) equipped with Image-Pro Insight 8.0 software (Media Cybernetics, Rockville, MD, USA).
8
Immunofluorescent Visualization of TβRII and IGF1R
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Cells were fixed in 100 % cold methanol for 10 min and permeabilized for 10 min in 0.3% Triton X-100 in PBS. After blocking with 5% normal Donkey serum (Ab7475, Abcam) for 1 h, cells were incubated overnight at 4 °C with following primary antibodies: TβRII (AF532, RD 1:100) and IGF1R (9750, CST 1:100). The cells were then incubated with Alexa Fluor 488 and 594 donkey anti-goat/rabbit IgG (H + L) (A-11055/R-37119, Life Technologies 1:500) for 2 h, and then mounted with Antifade Mounting medium with DAPI (VECTASHIELD). Fluorescence was examined by microscopy (Olympus IX-81).
9
Murine Sperm Immunofluorescence Characterization
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Murine SPZ collected from caput and cauda epididymis were dried on slides as above reported and fixed in 4% paraformaldehyde (sc-281692; Santa Cruz Biotechnology, Heidelberg, Germany) for 20 min at RT and then permeabilized with 0.2% Triton X-100 (X100; Sigma-Aldrich, Milano, Italy). Blocking was carried out with 10% of donkey serum (ab7475; Abcam, Cambridge, UK) for 30 min at RT and then cells were separately incubated with different primary antibodies [IZUMO1 (ab211623) from Abcam Cambridge, UK; PNA (L21409) from Invitrogen, Milano, Italy; TSSK6 (sc-514076) from Santa Cruz Biotechnology, Heidelberg, Germany] overnight at 4°C. Following three washes in DPBS (1X), a fluorescein isothiocyanate (FITC) conjugated was used as secondary antibody (711-095-152; Jackson ImmunoResearch, Cambridge, UK) for 1 h at 37°C. Nuclei were labeled with DAPI (D9542; Sigma-Aldrich, Milano, Italy), while F-actin was labeled with phalloidin (21834; Thermo Fisher Scientific, USA). All samples were analyzed under an optical microscope (Leica DM 5000 B + CTR 5000) with a UV lamp. Densitometric analysis of immunofluorescence was performed with ImageJ Software (version 1.53 g) and adjusted relatively to DAPI fluorescence intensity.
10
CUBIC Optical Clearing of Cortico-Striatal Assembloids
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