rabbit monoclonal anti ionized calcium binding adaptor molecule 1  (Danaher Inc)

Journal: Neural Regeneration Research

Article Title: High-dose dexamethasone regulates microglial polarization via the GR/JAK1/STAT3 signaling pathway after traumatic brain injury

doi: 10.4103/NRR.NRR-D-23-01772

Figure Lengend Snippet: Microglial activation in response to LPS and DEX treatment. (A) Representative immunofluorescence images showing that DEX treatment decreases M1 and M2 microglial polarization in cultured BV2 cells and primary microglia after exposure to LPS. Scale bars: 20 μm. Red represents Iba1, and green represents Arg1 or iNOS. (B) Counts of Arg1- and iNOS-positive cells normalized to Iba1-positive cells. * P < 0.05, vs . Control; # P < 0.05, vs . LPS (one-way analysis of variance with the least significant difference test). Data are presented as mean ± SD, n = 3. DAPI: 4′,6-Diamidino-2-phenylindole; DEX: dexamethasone; LPS: lipopolysaccharide.

Article Snippet: After blocking, cells were incubated with primary antibodies: rabbit monoclonal anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:200, Abcam, Cat# ab289370), mouse monoclonal anti-Arg1 (1:100, Abcam, Cat# ab239731), and mouse monoclonal anti-inducible nitric oxide synthase (iNOS) (1:500, Abcam, Cat# ab210823) for 24 hours at 4°C.

Techniques: Activation Assay, Immunofluorescence, Cell Culture, Control

Journal: Neural Regeneration Research

Article Title: Exercise preconditioning alleviates ischemia-induced memory deficits by increasing circulating adiponectin

doi: 10.4103/NRR.NRR-D-23-01101

Figure Lengend Snippet: Exercise pre-conditioning alleviates brain infarction and gliogenesis. (A) Experimental procedures. (B) Serially coronal sectioning views showing the brain infarction area from each group. (C) The calculation of infarction area based on TTC staining. One-way analysis of variance followed by Tukey’s post hoc test: F (2,7) = 15.76, P = 0.0026. n = 4, 3, and 3 mice in the Sham, MCAO and MCAO + Ex group, respectively. (D) Representative protein blotting bands of GFAP and Iba1 in prefrontal cortical tissue. (E) Quantification of relative protein expression level. Multiple t -test was used for between-group comparison. (F) Representative protein blotting bands of GFAP and Iba1 in hippocampal tissue. (G) Quantification of relative protein expression level. Multiple t -test was used for between-group comparison. (H) Immunofluorescent images for GFAP and Iba1 in dorsal hippocampus. (I) Relative fluorescent intensity. Multiple t -test was used for between-group comparison. n = 3 mice in E, G and I. All data are presented as mean ± SEM. DAPI: 4′,6-Diamidino-2-phenylindole; Ex: exercise pre-conditioning; GFAP: glial acidic fibrillary acidic proteins; Iba-1: ionized calciumbinding adapter molecule 1; MCAO: middle cerebral artery occlusion.

Article Snippet: Sections were then blocked using a commercially available blocking reagent (CAS-Block, Thermo Fisher Scientific, China Inc., Shanghai, China) at room temperature for 2 hours, and then incubated at 4°C for 36–48 hours with rabbit monoclonal anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:1000, FUJIFILM Wako Shibayagi, Shibukawa, Japan, Cat# 019-19741, RRID: AB_839504), goat polyclonal anti-glial fibrillary acidic protein (GFAP) (1:500, Thermo Fisher Scientific, Waltham, MA, USA, Cat# PA1-10004).

Techniques: Staining, Expressing, Comparison

Journal: Neural Regeneration Research

Article Title: Exercise preconditioning alleviates ischemia-induced memory deficits by increasing circulating adiponectin

doi: 10.4103/NRR.NRR-D-23-01101

Figure Lengend Snippet: Potentiation of the adiponectin pathway mimics exercise effects. (A) Quantification of blood adiponectin levels across all groups. One-way analysis of variance followed by Tukey’s post hoc test: F (2,12) = 6.839, P = 0.0104. (B) Experimental procedure. (C) Serial coronal sections showing brain infarct area (white) from each group (by 2,3,5-triphenyltetrazolium chloride [TTC] staining). (D) Calculation of infarct area based on TTC staining. One-way analysis of variance followed by Tukey’s post hoc test: F (2,12) = 8.272, P = 0.0055. n = 5 per group in A and D. (E) Representative protein bands for GFAP and Iba1 in hippocampal tissue. (F) Quantification of relative protein expression levels. Multiple t -test was used for intergroup comparison. n = 3 mice per group. (G) Immunofluorescent images for GFAP and Iba1 in dorsal hippocampus. All data are presented as mean ± SEM. DAPI: 4′,6-Diamidino-2-phenylindole; Ex: exercise pre-conditioning; GFAP: glial acidic fibrillary acidic proteins; Iba-1: ionized calcium-binding adapter molecule 1; MCAO: middle cerebral artery occlusion. Acute AdR: Acute AdipoRon + MCAO; Chronic AdR: chronic AdipoRon + MCAO.

Article Snippet: Sections were then blocked using a commercially available blocking reagent (CAS-Block, Thermo Fisher Scientific, China Inc., Shanghai, China) at room temperature for 2 hours, and then incubated at 4°C for 36–48 hours with rabbit monoclonal anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:1000, FUJIFILM Wako Shibayagi, Shibukawa, Japan, Cat# 019-19741, RRID: AB_839504), goat polyclonal anti-glial fibrillary acidic protein (GFAP) (1:500, Thermo Fisher Scientific, Waltham, MA, USA, Cat# PA1-10004).

Techniques: Staining, Expressing, Comparison, Binding Assay

Journal: Neural Regeneration Research

Article Title: TREM-1 mediates interaction between substantia nigra microglia and peripheral neutrophils

doi: 10.4103/1673-5374.385843

Figure Lengend Snippet: Upregulation of microglial TREM-1 in a PD model. (A, B) Western blotting and quantitative analysis of TREM-1 were conducted on days 1, 3, 5, and 7 after MPTP injection in the substantia nigra ( n = 3). (C, D) Western blotting and quantitative analysis of TREM-1 at 3, 6, 12, and 24 hours after MPP + treatment in BV2 cells ( n = 3). (E, F) Co-staining of TREM-1 (green, Alexa Fluor 488) and IBA-1 (red, Alexa Fluor 594) at day 7 post-MPTP administration and quantitative analysis of TREM-1 + cells. TREM-1 was overexpressed in microglia in the substantia nigra of the MPTP group compared with the saline group ( n = 4). Scale bars: 50 μm, 10 μm (enlarged image). Data are expressed as mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test [B, D] or Student’s t -test [F]). DAPI: 4,6-Diamidino-2-phenylindole; IBA1: ionized calcium bindingadaptor molecule-1; MPP + : 1-methyl-4-phenylpyridium; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; TREM-1: triggering receptor expressed on myeloid cell-1.

Article Snippet: Briefly, slices were preincubated with 5% donkey serum at 27°C for 60 minutes, followed by incubation overnight at 4°C using the following antibodies to investigate the effect of TREM-1 expression in microglia and neutrophils on dopaminergic neurons: rabbit anti-tyrosine hydroxylase (TH; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-14007, RRID: AB_671397), rabbit anti-ionized calcium binding adaptor molecule-1 (IBA1; 1:200, Cell Signaling Technology, Cat# 17198, RRID: AB_2820254), mouse anti-IBA1 (1:500, Servicebio, Wuhan, China, Cat# GB12105, RRID: AB_2922434), rabbit anti-myeloperoxidase (MPO; 1:200, Servicebio, Cat# GB11224, RRID: AB_2814688), rabbit anti-C-X-C ligand 1 (CXCL1; 1:200, Proteintech, Wuhan, China, Cat# 12335-1-AP, RRID: AB_2087568), rat anti-TREM-1 (1:200, Abcam, Cat# ab217161, RRID: AB_2938721), and rabbit anti-SYK (1:200, Cell Signaling Technology, Cat# 13198, RRID: AB_2687924).

Techniques: Western Blot, Injection, Staining, Saline, Comparison

Journal: Neural Regeneration Research

Article Title: TREM-1 mediates interaction between substantia nigra microglia and peripheral neutrophils

doi: 10.4103/1673-5374.385843

Figure Lengend Snippet: TREM-1 knockout suppresses chemokine production and neutrophil invasion. (A–D) Relative mRNA expression of CXCL1 , CXCL2 , and CXCL8 , and the adhesion molecule, ICAM-1 ( n = 3). (E, F) Co-staining for visualization of IBA1 (red, Alexa Fluor 594) and CXCL1 (green, Alexa Fluor 488) on day 7 after MPTP administration and quantitative analysis of IBA1 + CXCL1 + cells Downregulation of CXCL1 expression in microglia of substantia nigra in the MPTP group after TREM-1 knockout ( n = 4). (G, H) Immunofluorescence and quantification of MPO (red, Alexa Fluor 594). MPO expression was markedly reduced after TREM-1 knockdown in the PD mouse model ( n = 4). Scale bars: 50 μm, 10 μm (enlarged image). Data are expressed as mean ± SEM. ** P < 0.01, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test [A–D, F] or Student’s t -test [H]). CXCL1: C-X-C ligand 1; CXCL2: C-X-C ligand 2; CXCL8: C-X-C ligand 8; DAPI: 4,6-diamidino-2-phenylindole; IBA1: ionized calcium binding adaptor molecule-1; ICAM-1: intracellular adhesion molecule-1; MPO: myeloperoxidase; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; TREM-1 –/– : TREM-1 knockout; WT: wild type.

Article Snippet: Briefly, slices were preincubated with 5% donkey serum at 27°C for 60 minutes, followed by incubation overnight at 4°C using the following antibodies to investigate the effect of TREM-1 expression in microglia and neutrophils on dopaminergic neurons: rabbit anti-tyrosine hydroxylase (TH; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-14007, RRID: AB_671397), rabbit anti-ionized calcium binding adaptor molecule-1 (IBA1; 1:200, Cell Signaling Technology, Cat# 17198, RRID: AB_2820254), mouse anti-IBA1 (1:500, Servicebio, Wuhan, China, Cat# GB12105, RRID: AB_2922434), rabbit anti-myeloperoxidase (MPO; 1:200, Servicebio, Cat# GB11224, RRID: AB_2814688), rabbit anti-C-X-C ligand 1 (CXCL1; 1:200, Proteintech, Wuhan, China, Cat# 12335-1-AP, RRID: AB_2087568), rat anti-TREM-1 (1:200, Abcam, Cat# ab217161, RRID: AB_2938721), and rabbit anti-SYK (1:200, Cell Signaling Technology, Cat# 13198, RRID: AB_2687924).

Techniques: Knock-Out, Expressing, Staining, Immunofluorescence, Knockdown, Comparison, Binding Assay