Journal: Glia

Article Title: Activated microglia do not increase 18 kDa translocator protein ( TSPO ) expression in the multiple sclerosis brain

doi: 10.1002/glia.24052

Figure Lengend Snippet: Antibodies for immunohistochemistry

Article Snippet: CD68 , Rabbit , Atlas Antibody , HPA048982 , 1:4000.

Techniques:

Journal: bioRxiv

Article Title: Association of cerebellar inflammation and neurodegeneration in a novel spinocerebellar ataxia type 13 mouse model

doi: 10.1101/2024.10.28.620701

Figure Lengend Snippet: To examine macrophage activation and Purkinje cells loss in the cerebellum, the location and protein levels of CD 68 (macrophage marker) and Calbindin (Purkinje cell marker) were examined in the cerebella of mice at different time points. Half of cerebella of mice were fixed for immunofluorescence staining, other half of the cerebella were fresh frozen for western blot analysis. (A) Images representatives of anti-Calbindin and anti-CD68 at the ages of 1,3, and 6 months. (B) Quantitative analysis of positive cells number of immunofluorescence staining signal of CD68. (C) Quantitative analysis of positive cells number of immunofluorescence staining signal of Calbindin. (D) Western blot images of CD68 and Calbindin. (E) Quantitative analysis of western blot of CD68 and Calbindin. Note: Data was presented as mean ± SEM and unpaired t-test or one-way ANOVA Turkey multiple comparison test was used for quantitative analysis in GraphPad Prism 10. * p <0.05, **** p <0.0001.

Article Snippet: In the last step slides were incubated overnight the following conjugated antibodies: anti-Calbindin 488 (Cell signaling, #13176, 1:100), anti-Glial Fibrillary Acidic Protein (GFAP) Alexa Fluor 488 (Invitrogen, A-21294, 1:200), Ionized calcium binding adaptor molecule 1 (Iba1) Alexa Fluor 488 (Cell Signaling, #20825, 1:100), and CD68 Alexa Fluor 488 (Cell Signaling, #51644, 1:200).

Techniques: Activation Assay, Marker, Immunofluorescence, Staining, Western Blot, Comparison

Journal: bioRxiv

Article Title: Association of cerebellar inflammation and neurodegeneration in a novel spinocerebellar ataxia type 13 mouse model

doi: 10.1101/2024.10.28.620701

Figure Lengend Snippet: To examine EGFR expression in macrophage, the colocalization of CD68 and EGFR were investigated in the cerebella of mice at different time points. Images representatives of anti-CD68 and anti-EGFR at the ages of 1,3, and 6 months.

Article Snippet: In the last step slides were incubated overnight the following conjugated antibodies: anti-Calbindin 488 (Cell signaling, #13176, 1:100), anti-Glial Fibrillary Acidic Protein (GFAP) Alexa Fluor 488 (Invitrogen, A-21294, 1:200), Ionized calcium binding adaptor molecule 1 (Iba1) Alexa Fluor 488 (Cell Signaling, #20825, 1:100), and CD68 Alexa Fluor 488 (Cell Signaling, #51644, 1:200).

Techniques: Expressing

Journal: bioRxiv

Article Title: Association of cerebellar inflammation and neurodegeneration in a novel spinocerebellar ataxia type 13 mouse model

doi: 10.1101/2024.10.28.620701

Figure Lengend Snippet: To assess the expression of phosphorylated (Tyr1068) EGFR (pEGFR) in macrophage, the location and protein levels of pEGFR and CD68 were examined in the cerebella of mice at different time points. Half of cerebella of mice were fixed for immunofluorescence staining, other half of the cerebella were fresh frozen for western blot analysis. (A) Images representatives of anti- pEGFR and anti-CD68 at the ages of 1,3, and 6 months. (B) Quantitative analysis of positive cells number of immunofluorescence staining signal of pEGFR (D) Western blot images of pEGFR. (E) Quantitative analysis of western blot of pEGFR. Note: Data was presented as mean ± SEM and unpaired t-test or one-way ANOVA Turkey multiple comparison test was used for quantitative analysis in GraphPad Prism 10. * p <0.05, **** p <0.0001.

Article Snippet: In the last step slides were incubated overnight the following conjugated antibodies: anti-Calbindin 488 (Cell signaling, #13176, 1:100), anti-Glial Fibrillary Acidic Protein (GFAP) Alexa Fluor 488 (Invitrogen, A-21294, 1:200), Ionized calcium binding adaptor molecule 1 (Iba1) Alexa Fluor 488 (Cell Signaling, #20825, 1:100), and CD68 Alexa Fluor 488 (Cell Signaling, #51644, 1:200).

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Comparison

Journal: bioRxiv

Article Title: Association of cerebellar inflammation and neurodegeneration in a novel spinocerebellar ataxia type 13 mouse model

doi: 10.1101/2024.10.28.620701

Figure Lengend Snippet: Pearson correlation was analyzed among EGFR/pEGFR, CD68, GFAP, Iba-1 and Calbindin according to our quantitative expression data. (A) Correlation between EGFR positive cell number and Calbindin positive cell number. (B) Correlation between pEGFR positive cell number and Calbindin positive cell number. (C) Correlation between CD68 positive cell number and Calbindin positive cell number. (D) Correlation between EGFR positive cell number and CD68 positive cell number. (E) Correlation between pEGFR positive cell number and CD68 positive cell number. (F) Correlation between pEGFR positive cell number and the intensity of GFAP positive signal.

Article Snippet: In the last step slides were incubated overnight the following conjugated antibodies: anti-Calbindin 488 (Cell signaling, #13176, 1:100), anti-Glial Fibrillary Acidic Protein (GFAP) Alexa Fluor 488 (Invitrogen, A-21294, 1:200), Ionized calcium binding adaptor molecule 1 (Iba1) Alexa Fluor 488 (Cell Signaling, #20825, 1:100), and CD68 Alexa Fluor 488 (Cell Signaling, #51644, 1:200).

Techniques: Expressing

Journal: Antioxidants

Article Title: Genipin Attenuates Diabetic Cognitive Impairment by Reducing Lipid Accumulation and Promoting Mitochondrial Fusion via FABP4/Mfn1 Signaling in Microglia

doi: 10.3390/antiox12010074

Figure Lengend Snippet: GEN shifted microglia polarization and inhibited lipid accumulation in LPS/HG/PA-induced HMC3 cells. The HMC3 cells were treated with GEN (5, 10, 20 μM) for 4 h and then stimulated with LPS/HG/PA for 12 h. The supernatant concentrations of TNF-α ( A ), IL-6 ( B ), IL-4 ( C ) and IL-10 ( D ) were examined by ELISA (n = 4). The mRNA expressions of iNOS ( E ), CCL2 ( F ), ARG1 ( G ) and YM1 ( H ) in cells were assessed by PCR (n = 4). The population of CD68-positive and CD206-positive cells were measured by flow cytometry ( I ). The expressions of FABP4, p-NF-κB and NF-κB were detected by Western blot ( J – L ) (n = 3). The nucleus translocation of NF-κB was visualized by immunofluorescence staining under laser confocal microscope. The scale bar equaled 5 μm ( M ). The lipid accumulation was observed by oil red O staining. The scale bar equaled 50 μm ( N ). The analysis of oil red O staining ( O ). The mRNA expressions of fatty acid β-oxidation genes including ACOX1 , ACAA2 and ECHS1 were measured by PCR ( P ). The mRNA expressions of fatty acid uptake genes including SLC27A1 and PPARα were measured by PCR ( Q ). The mRNA expressions of fatty acid synthesis genes including FASN and ACLY were measured by PCR ( R ). The results are expressed as means ± SDs. ### p < 0.001 compared with control group. * p < 0.05, ** p < 0.01 compared with LPS/HG/PA group or the other group. ns means not significant.

Article Snippet: Mfn2 (#9482S), Drp1 (#8570S), p-Nuclear Factor Kappa B (NF-κB) (#3033), NF-κB (#8242), CD68 (#91882), p47phox (#4312), β-actin (#4970), Na, K-ATPase (#3010), ubiquitin (#3936), Flag (#8146), Flag (#14793), HA (#3724), Myc (#2276), V5 (#80076), mouse anti-rabbit IgG (Conformation Specific, #5127), rabbit anti-mouse IgG (Light Chain Specific, #58802) and mouse anti-rabbit IgG (Light-Chain Specific, #93702) antibodies were provided by Cell Signaling Technology (Danvers, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Western Blot, Translocation Assay, Immunofluorescence, Staining, Microscopy, Control

Journal: International Journal of Molecular Sciences

Article Title: The Activation of the Microglial NLRP3 Inflammasome Is Involved in Tuberous Sclerosis Complex-Related Neuroinflammation

doi: 10.3390/ijms26157244

Figure Lengend Snippet: Detection of HMC3 cell activation and ROS levels. ( A ) The mRNA level of AIF1 was elevated in the TSC2 KD group. ( B , C ) AIF1 fluorescence intensity was greater in TSC2 KD HMC3 cells than in control HMC3 cells. ( D ) The mRNA level of CD68 was elevated in the TSC2 KD group. ( E , F ) The CD68 fluorescence intensity of TSC2 KD HMC3 cells was greater than that of control cells. ( G , H ) The intensity of ROS fluorescence in TSC2 KD HMC3 cells was greater than that in control cells. DHE: Dihydroethidium; N = 3; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The cells were subsequently treated with reagents containing 0.5% Triton X-100 and 5% fetal bovine serum for 30 min. Anti-AIF1 primary antibody (recombinant rabbit monoclonal, 1:500; HUABIO, Hangzhou, China) and anti-CD68 primary antibody (mouse monoclonal, 1:500; Proteintech, Wuhan, China) were incubated at 4 °C overnight.

Techniques: Activation Assay, Fluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: The Activation of the Microglial NLRP3 Inflammasome Is Involved in Tuberous Sclerosis Complex-Related Neuroinflammation

doi: 10.3390/ijms26157244

Figure Lengend Snippet: The effect of rapamycin intervention on HMC3 cells was detected. ( A – C ) Immunofluorescence assays revealed that AIF1 expression was downregulated in the TSC2 KD + Rapa group and the control + Rapa group and that CD68 expression was downregulated in the TSC2 KD + Rapa group. ( D ) RT-qPCR revealed a decrease in AIF1 mRNA levels in the TSC2 KD + Rapa group and the control + Rapa group and a decrease in CD68 mRNA levels in the TSC2 KD + Rapa group. ( E , F ) The level of ROS was significantly lower in the TSC2 KD + Rapa group than in the control + Rapa group. Rapa: rapamycin; N = 3; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The cells were subsequently treated with reagents containing 0.5% Triton X-100 and 5% fetal bovine serum for 30 min. Anti-AIF1 primary antibody (recombinant rabbit monoclonal, 1:500; HUABIO, Hangzhou, China) and anti-CD68 primary antibody (mouse monoclonal, 1:500; Proteintech, Wuhan, China) were incubated at 4 °C overnight.

Techniques: Immunofluorescence, Expressing, Control, Quantitative RT-PCR