Four to eight weeks after transplantation, rats were transcardially perfused with 0.1 M phosphate buffered saline, followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were dissected (Figure 1D ) and post-fixed in the same solution for 12 h and then transferred to a 30% sucrose solution for 24 h. Brains were frozen in embedding matrix using dry ice and stored at −20°C before being sectioned on a cryostat at 40-μm thickness. Free-floating sections were stored in 0.02% sodium aside in phosphate buffered saline prior to immunohistochemistry. Samples were stained with 4′,6-diamidino-2-phenylindole (DAPI) to mark neuronal nuclei and GFP to confirm the presence of transplanted hNSCs. Samples were also assessed with the following primary antibodies: NeuN (Millipore MAB377), DCX (Millipore AB2253), GFAP (Dako Z0334), and IBA-1(Millipore MABN92). Fluorescent images were observed on a confocal microscope (OLYMPUS BX51, Olympus Optical Co., Ltd., Tokyo, Japan).
Mabn92
Manufactured by Merck Group
Sourced in United States, Germany
MABN92 is a laboratory instrument designed for conducting various scientific experiments and analyses. Its core function is to provide precise and reliable measurements of physical and chemical properties of samples. The details of its intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Mab377, by Merck Group (2 mentions)
Rabbit anti gfap, by Merck Group (2 mentions)
Sc 13914, by Santa Cruz Biotechnology (2 mentions)
Z0334, by Agilent Technologies (2 mentions)
Z0334, by Merck Group (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Rpn4301, by Cytiva (1 mentions)
Rpn4201, by Cytiva (1 mentions)
Cw0096m, by CWBIO (1 mentions)
Dylight 549 goat anti rabbit igg, by Abbkine (1 mentions)
Ab18258, by Abcam (1 mentions)
Mab360, by Merck Group (1 mentions)
Ab109373, by Abcam (1 mentions)
Ab183781, by Abcam (1 mentions)
Ab30760, by Abcam (1 mentions)
Ab84036, by Abcam (1 mentions)
Ab104224, by Abcam (1 mentions)
Dylight 488 goat anti mouse igg, by Abbkine (1 mentions)
Ab199000, by Abcam (1 mentions)
Ab80437, by Abcam (1 mentions)
23 protocols using mabn92
1
Histological Analysis of Transplanted hNSCs
2
Immunofluorescence Staining of Spinal Cord
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Mice were perfused with PBS followed by 10% formalin. The separated spinal cords were post-fixed overnight, cryoprotected, and incubated with 10%, 20%, and 30% sucrose, respectively, for 12 hours. Spinal cords were cut into 40 μm-thick coronal sections. Three sections per mice were selected for later immunofluorescence. Nonspecific binding sites were blocked using 0.3% bovine serum albumin (BSA) with 0.3% Triton X-100 in PBS for 1 hour at room temperature. After blocking, the sections were incubated with mouse anti-GFAP (marker of astrocyte; 1:500; Invitrogen; 14-9892-82) or mouse anti-Iba-1 (marker of microglia; 1:500; Millipore; MABN92) for 72 hours at 4°C. The sections were then incubated with secondary antibody of Alexa Fluor 488 donkey anti-rabbit IgG (A21206, 1:500 dilution) diluted in PBS with 0.3% Triton X-100 for 1 hour at room temperature. Then fluorescence mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) was finally added. The sections were observed under an Olympus FV-1000 laser confocal scanning microscope (Olympus Corporation, Shinjuku, Tokyo, Japan) equipped with a plan apochromatic × 60 oil immersion objective.
3
Antibody Characterization for Iron Metabolism
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The following antibodies were used: β-actin antibody (cw0096m, CWbio, China), FPN1 antibody (MTP11-S, ADI, USA), DMT1 (+IRE) antibody (NRAMP21-S, ADI, USA), 4-HNE antibody (HNE-11S, ADI, USA), TfR1 antibody (ab84036, Abcam, USA), L-ferritin antibody (ab109373, Abcam, USA), H-ferritin antibody (ab183781, Abcam, USA), hepcidin antibody (ab30760, Abcam, USA), Aβ22–35 antibody (A3356, Sigma, USA), phospho-p38 (p-p38) antibody (4511S, CST, USA), p38 antibody (8690S, CST, USA), phospho-ERK (p-ERK) antibody (9102S, CST, USA), ERK antibody (4372S, CST, USA), Bcl-2 antibody (12789-1-AP, Proteintech, China), Bax antibody (50599-2-Ig, Proteintech, China), GFAP antibody (MAB360, Millipore, USA), Iba1 antibody (MABN92, Millipore, USA), NeuN (ab104224, Abcam, USA), CD31 (77699T, CST, USA), PSD-95 (ab18258, Abcam, USA), anti-rabbit IgG (RPN4301, Amersham, UK), anti-mouse IgG (RPN4201, Amersham, UK), DyLight 488 goat anti-mouse IgG (A23210, Abbkine, USA), DyLight 549 goat anti-rabbit IgG (A23320, Abbkine, USA), and DyLight 549 goat anti-mouse IgG (A23310, Abbkine, USA).
4
Comprehensive Immunohistochemical Profiling
5
Western Blot Analysis of Spinal Cord
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The L4-L6 spinal cord segments were rapidly extracted from the anesthetized rats. The tissues were homogenized in lysis buffer (Bio-Rad Laboratories, Hercules, CA, USA) which contains a mixture of protease inhibitors and phenylmethylsulfonyl fluoride (Roche Diagnostics, Basel, Switzerland). Equivalent amounts of protein (10 μl) were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad Laboratories), and transferred onto PVDF membranes (Millipore). The membranes were incubated with the primary antibodies overnight at 4°C, followed by HRP-conjugated secondary antibodies for 2 h (1:1,000; #7074 and #7076; Cell Signaling Technology). The secondary antibody signal was detected by enhanced chemiluminescence (Millipore), and captured using the ChemiDoc XRS system (Bio-Rad Laboratories). The following primary antibodies were used: rabbit anti-p-JNK (1:1,000; ab4821), rabbit anti-JNK (1:1,000; ab85139), rabbit anti-PDE4B (1:1,500; ab14611) (all from Abcam), mouse anti-GFAP (1:2,000; #3670; Cell Signaling Technology), goat anti-IBA1 (1:1,000; MABN92; Millipore) and mouse anti-β-actin (1:10,000; A1978; Sigma-Aldrich).
6
Immunofluorescence Analysis of Spinal Cord Samples
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The rats were deeply anesthetised with an overdose of chloral hydrate (10%, 0.3 ml/100 g) and transcardially perfused with 200 ml of 0.01 M PBS (pH 7.4), followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The L4–L6 spinal cord segments with the spinal dorsal horn (SDH) were harvested and dehydrated in 30% sucrose at 4°C. Transverse spinal sections (30-μm-thick) were then cut using a cryostat (CM3050S; Leica, Wetzlar, Germany). For double immunofluorescence, the sections were incubated with rabbit anti-c-Fos (1:500; ab209794; Abcam, Cambridge, MA, USA), mouse anti-glial fibrillary acidic protein (GFAP) (1:400; #3670; Cell Signaling Technology, Danvers, MA, USA) and goat anti-IBA-1 (1:500; MABN92; Millipore, Billerica, MA, USA) antibodies, followed by FITC-conjugated secondary antibodies [Alexa Fluor 594 donkey anti-rabbit IgG (A21207), Alexa Fluor 488 donkey anti-mouse IgG (A21202) and Alexa Fluor 488 donkey anti-goat IgG (A11055); 1:500; all from Invitrogen, Carlsbad, CA, USA] for 2 h at room temperature. The stained sections were observed and captured under a confocal laserscanning microscope (FV1000; Olympus, Tokyo, Japan).
7
Immunostaining of mouse tissue samples
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Primary antibodies used in the experiments were rat anti-PECAM1 (BD Biosciences #553370, RRID:AB_394816), rat anti-LYVE1 (Thermo Fisher Scientific #14-0443-82, RRID:AB_1633414), chicken anti-GFP (Aves Labs #GFP-1010, RRID:AB_2307313), rat anti-CD11b (Biolegend #101202, RRID:AB_312785), rabbit anti-RFP (Rockland #600-401-379, RRID:AB_2209751), mouse anti-Iba1 (Millipore #MABN92, RRID:AB_10917271), rabbit anti-Olig2 (Millipore #AB9610, RRID:AB_10141047), goat anti-IRF8 (Santa Cruz #sc-6058, RRID:AB_649510), rabbit anti-PU.1 (Cell Signaling #2258, RRID:AB_10693421), rabbit anti-Ki67 (Millipore #AB9260, RRID:AB_2142366) and mouse anti-p-IκBα (Cell Signaling #9246, RRID:AB_2267145). Alexa Fluor-conjugated secondary antibodies were from Thermo Fisher Scientific.
8
Immunohistochemical Detection of Iba1 and 4-HNE
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Tissue slices were washed three times with PBS. Antigen retrieval was performed in a microwave oven for 10 min in 10 mM citrate buffer (pH 6.0). After blocking for 1 h with normal goat serum prepared in PBS, the slices were incubated overnight at 4 °C with a mouse anti-Iba1 monoclonal antibody (1:200; #MABN92, Millipore Corporation, Temecula, CA) or a rabbit anti-4-hydroxynonenal (4-HNE) polyclonal antibody (1:400; #HNE13-M, Alpha Diagnostic International, San Antonio, TX, USA). The slides were then washed three times for 5 min with PBS. The following secondary antibodies were used for 50 min incubations at 37 °C: DyLight 549-conjugated goat anti‐rabbit IgG (1:200; #A23320, Abbkine Scientific Co., Ltd., Wuhan, China) and DyLight 488-conjugated goat anti‐mouse IgG (1:200; #A23210, Abbkine Scientific Co., Ltd., Wuhan, China). After washing and mounting, the sections were analyzed with an Olympus FV3000 confocal laser scanning microscope.
9
Analyzing Retinal Cell Populations and Dendrimer Uptake
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PF-PBS post-fixed retina, lamina and ON frozen sections were analyzed for fluorescent dendrimer signal (D-Cy5), as well as for RGCs (goat anti human Brn3a; Santa Cruz Sc-31984; 1:500 concentration), total inflammatory cells (mouse anti-human IBA1; Millipore MABN92; 1:500 concentration), and astrocytes (glial acidic fibrillary protein [rabbit anti-pig GFAP; Sigma-Aldrich clone GA5; 1:1000 concentration] and aldolase dehydrogenase 1L1 [rabbit anti-mouse Aldh1L1; Abcam ab87117]; 1:1000 concentration). We performed antigen recovery using citrate buffer. Primary antibodies made in different species (mouse antihuman IBA1 and rabbit antimouse Aldh1L1) enabled us to colocalize both Aldh1L1 and IBA1 without difficulty, using fluorescently labeled donkey anti-rabbit (Cy2) and donkey anti-mouse (Cy3) secondary antibodies. Histological analysis was performed using a 4-channel confocal microscope (Olympus E900).
10
Immunofluorescent Analysis of Microglia and TREM2
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Astrocytes or microglia were seeded at 2 × 105 cells/well in 18 mm diameter coverslips. Once the culture reached 90% confluence, the cells were fixed in PBS containing 4% paraformaldehyde (PFA) for 15 min. Cells were permeabilized for 5 min at RT in 0.25% Triton X-100 in PBS, washed twice with PBS, and incubated for 1 h at 37 °C in PBS containing 5% goat, 5% horse serum, and 2% fish gel (blocking solution). Cells were then incubated overnight at 4 °C in primary antibody diluted 1:100 in blocking solution with anti-IBA1 (MABN92, Millipore) and anti-TREM2 (AF1729, R&D). After incubation, cells were washed with PBS, then incubated with Alexa 488-conjugated goat anti-mouse antibody (1:100, Invitrogen) and Alexa 568-conjugated goat anti-rabbit antibody (1:100, Invitrogen) for 1 h at RT, then washed with PBS and mounted with Vectashield mounting medium with DAPI (Vector Laboratories). Samples were examined using a Nikon A1-R laser scanning confocal microscope coupled with Nikon AR software for orthogonal images of reconstructed three-dimensional views. At least 5–10 cells were analyzed from each image. Eight to ten images were used for each experiment, and three independent culture experiments were performed.
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