Cryosections 14 μm thick were prepared according to the previous publication (Bae et al., 2009 (link)). The sections were immunostained as previously described (Bae et al., 2009 (link); Kani et al., 2010 (link)). The following antibodies were used: anti-GFP (1:1000, rat, Nacalai Tesque, catalog #04404-84, RRID: AB 10013361 or 1:1000, rabbit, MBL International, catalog #598, RRID: AB_591816 ) for BoTxBLC-GFP, anti-Neurod1 (1:400, mouse, ascites; Kani et al., 2010 (link)), and anti-parvalbumin 7 (1:1000, mouse monoclonal, ascites; Bae et al., 2009 (link)). The following secondary antibodies were used: Alexa Fluor 488 goat anti-rat (H + L, Invitrogen, Thermo Fisher Scientific, catalog #A11006, RRID: AB_2534074 ), CF488A anti-rabbit (H + L, Biotium Inc., catalog #20019, RRID: AB_10583180 ), and Alexa Fluor 568 goat anti-mouse IgG (H + L, Invitrogen, catalog #A11031, RRID: AB_144696 ). An LSM700 confocal laser-scanning microscope was used to obtain fluorescence images. GFP+ areas in the granular layer (GL) were measured by ImageJ software (https://imagej.nih.gov/ij/ ) (Table 1 ).
Alexa fluor 488 goat anti
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States, China
Alexa Fluor 488 goat anti is a fluorescent labeling reagent used for detection and visualization applications in research. It is a conjugate of the Alexa Fluor 488 dye and a goat-derived antibody. The core function of this product is to provide a fluorescent label that can be used to detect and localize target molecules in samples.
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Lab products found in correlation
Lsm 800, by Zeiss (2 mentions)
Alexa fluor 647 goat, by Thermo Fisher Scientific (1 mentions)
Mab374, by Cell Signaling Technology (1 mentions)
Ha tag, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Abn78a4, by Merck Group (1 mentions)
Olig2, by Abcam (1 mentions)
Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Clsm780 microscope, by Zeiss (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Alexa fluor594 goat anti, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Merck Group (1 mentions)
H 1200, by Vector Laboratories (1 mentions)
Laser confocal microscope, by Olympus (1 mentions)
Sc 32251, by Santa Cruz Biotechnology (1 mentions)
Ab34710, by Abcam (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
A11032, by Thermo Fisher Scientific (1 mentions)
A27034, by Thermo Fisher Scientific (1 mentions)
9 protocols using alexa fluor 488 goat anti
1
Immunostaining of Cryosectioned Brain Tissue
2
Immunoblotting for SUMOylated Proteins
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Western blots (WB) and peptide arrays (PA) were immunoblotted for various proteins. The following antibodies were used: GAPDH (1 : 5000 WB; Millipore, Burlington, MA, USA; MAB374), HA tag (1 : 500 IF, 1 : 1000 WB; Cell Signalling Technologies, Danvers, MA, USA; 2367), SUMO‐TnI (1 : 100 PA, WB, custom antibody produced in rabbits against SUMOylated synthetic peptide N‐HLKQVKKEDTEK‐C; Badrilla, Leeds, UK), SUMO1 (1 : 1000 PA, WB, Enzo BML‐PW9465‐0025), TnI (1 : 500 IF, 1 : 1000 WB; Abcam, Waltham, MA, USA; ab27003), Donkey anti‐mouse fluorescent secondary (1 : 5000 WB; Licor, Lincoln, NE, USA; 925–68 072), Donkey anti‐rabbit fluorescent secondary (1 : 5000 WB; Licor 925–68 073), Goat anti‐rabbit HRP secondary (1 : 5000 PA, WB; Sigma, Welwyn Garden City, UK; A6154), Alexa Fluor® 488 goat anti‐mouse (1 : 500 IF; Invitrogen, Waltham, MA, USA; A21071), and Alexa Fluor® 647 goat anti‐rabbit (1 : 500 IF; Invitrogen A21121).
3
Immunohistochemical Profiling of Prefrontal Cortex
4
Immunofluorescence Staining of Brain Tissue
5
Immunofluorescence Analysis of Pancreatic Tissue
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Immunofluorescence analysis of α-smooth muscle actin (α-SMA) and collagen I was performed using the pancreatic tissue. The pancreatic tissues were fixed in a 10% neutral-buffered-formalin solution for 24 h, embedded within optimal cutting temperature compound, and cut into 9 μm sections. The tissues were incubated with primary antibodies against α-SMA (1:500; sc-32251; Santa Cruz Biotechnology, Texas, Dallas, United States) and collagen I (1:250; ab34710; Abcam, Cambridge, United Kingdom) overnight at 4°C, followed by fluorescence-labeled secondary antibodies Alexa Fluor® 594 goat anti-mouse (1:2,000; A11032; Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States) and Alexa Fluor® 488 goat anti-rabbit (1:2,000; A27034; Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States) at RT for 2 h. Nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI; 5 ng/ml; D1306; Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States) at room temperature for 5 min. The stained sections were visualized using a confocal laser microscope (Olympus Corporation, Tokyo, Japan).
6
Immunostaining Procedure for NLRP3, KAT5, GOLGA4, TGN38
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For immunostaining, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS, then the cells were incubated with primary antibodies(anti-NLRP3 antibody (1:100), anti-KAT5 antibody (1:100), anti-GOLGA4(1:200) or anti-TGN38(1:200)) followed by staining with secondary antibody(DyLight 488-labeled secondary antibody (Invitrogen)(1:50),Alexa Fluor 594-conjugated secondary Ab (Invitrogen) (1:50),Alexa Fluor® 488 Goat anti-mouse IgG (minimal x-reactivity) Antibody(1:100) or Cy3–conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (1:100)), while nuclei were stained with DAPI containing mounting medium (Beyotime). To better preserve the dTGN structures in COS-7 cells, 0.01% Triton X-100 and 0.1% saponin were respectively in place of 0.1% Triton X-100 in permeabilization step for immunostaining of GOLGA4 and TGN38. Fluorescence images for fixed cells were taken with confocal fluorescence microscope (SpinSR10; Olympus) and (LSM800; ZEISS).
7
SARS-CoV-2 Spike Protein Detection
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During and following longer-term treatment assays, chamber slides were fixed with methanol and stained with first antibody SARS-CoV-2 spike chimeric monoclonal antibody (Sino Biological #40150-D004, Beijing, China) at 1:1000 dilution and second antibody Alexa Fluor 488 goat anti-human immunoglobulin G (IgG) (H + L) (Invitrogen #A-11013, Paisley, UK) at 1:500 dilution combined with Hoechst 33342 (Invitrogen, Paisley, UK) at 1:1000 dilution. Fluorescence microscopy (ZEISS Axio Vert.A1, Jena, Germany) was used to evaluate the percentages of SARS-CoV-2 spike-positive cells, using the following designations: 0% positive cells (no cells infected), single positive cells, and 10 to 90% positive cells, in steps of 10%.
8
Immunohistochemical Analysis of Stem Cell Markers
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Patient samples (LV1221) were purchased from US Biomax (Rockville) and analyzed for the expression of TIPRL, LC3, CD133 and CD46 as the following; briefly, de-paraffinization was performed by immersing each slide in the order of xylene, as well as 100, 95, and 70% ethanol (Merck, 1.00983.1011) for five minutes each. Then the slides were subjected to a pre-heated sodium citrate buffer (0.01 M, pH 6.0; Merck, S4641) for 15 minutes to retrieve antigens. After that, the slides were incubated with antibodies against TIPRL (1:100; Bethyl laboratories, A300-663A), LC3 (1:100; Merck, L7543), CD133 (1:100; NOVUS, NBP2-37741) and CD46 (1:200; SCBT, sc-52647) overnight. The slides were then reacted with Alexa Fluor 633 goat anti-rabbit (1:100, TIPRL; Thermo Fisher Scientific, A11079), Alexa Fluor 568 goat anti-rabbit (1:100, LC3; Thermo Fisher Scientific, A21071) and Alexa Fluor 488 goat anti-mouse (1:100, CD133 and CD46; Thermo Fisher Scientific, T7458), respectively. Confocal observation (ZEISS LSM 800) was followed by quantification of each expression (Supplementary Table 2 ) using the Carl Zeiss LSM Image program (ZEN 2.3 lite) according to a previous study29 (link).
Patient information provided by US Biomax (Rockville) was used for this study (Supplementary Tables1 and 3 ).
Patient information provided by US Biomax (Rockville) was used for this study (Supplementary Tables
9
Immunostaining of Fluorescent Proteins in Dissected Brains
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Brain dissections, fixation, and immunostaining were performed as described.39 (link),98 (link) To visualize native GFP, tdTomato, and mVenus, we followed our previous adult brain protocol.100 (link) In short, dissected brains were fixed in 4 % [w/v] paraformaldehyde in PBS (1.86 mM NaH2PO4, 8.41 mM Na2HPO4, 175 mM NaCl) for 20 minutes at room temperature. Samples were washed for 3×20 minutes in PBS containing 0.3% (v/v) Triton-X-100 (PBT) and blocked with 5% normal goat serum for at least 30 minutes. Samples were then incubated for 2 days with primary antibodies. After being washed three times with PBT, samples were incubated for 2 days with secondary antibodies in PBT followed by additional washing in PBT and embedding in Vectashield. Primary antibodies used: rabbit anti-GFP (Thermo Fisher Scientific A-11122, 1:1000), mouse anti-GFP (Abcam ab1218, 1:250), rabbit anti-RFP (Abcam ab62341, 1:250), mouse anti-Bruchpilot (DSHB, 1:50). Secondary antibodies used: Alexa Fluor 488 goat anti-mouse (Abcam ab150113, 1:500), Alexa Fluor 488 goat anti-rabbit (Thermo Fisher Scientific A-11034, 1:500), Alexa Fluor 568 goat anti-rabbit (Abcam ab175471, 1:500), Alexa Fluor 647 goat anti-mouse (Abcam ab150115, 1:500). Images were collected on a Leica TCS SP5 (LAS AF software v2.7.3.9723) with HCX PL APO lambda blue 20.0x0.70 IMM UV objective and processed in Fiji/ImageJ.94 (link)
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