PA5-21274

antibody from Invitrogen Antibodies

Targeting: AIF1 AIF-1, Em:AF129756.17, IBA1, IRT-1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-21274

Provider

Invitrogen Antibodies

Product name

IBA1 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

Recommended positive controls: K562, THP-1, HL-60, mouse brain, mouse liver, rat liver, Mouse brain. Predicted reactivity: Mouse (100%), Rat (100%), Pig (100%), Rhesus Monkey (100%). Store product as a concentrated solution. Centrifuge briefly prior to opening the vial.

Reactivity

Human, Mouse, Rat

Host

Rabbit

Isotype

IgG

Vial size

100 μL

Concentration

1.0 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

References

Submitted references

In Utero Exposure to Zika Virus Results in sex-Specific Memory Deficits and Neurological Alterations in Adult Mice.

Andrade TA, Fahel JS, de Souza JM, Terra AC, Souza DG, Costa VV, Teixeira MM, Bloise E, Ribeiro FM
ASN neuro 2022 Jan-Dec;14:17590914221121257

---

METTL3-dependent RNA m(6)A dysregulation contributes to neurodegeneration in Alzheimer's disease through aberrant cell cycle events.

Zhao F, Xu Y, Gao S, Qin L, Austria Q, Siedlak SL, Pajdzik K, Dai Q, He C, Wang W, O'Donnell JM, Tang B, Zhu X
Molecular neurodegeneration 2021 Sep 30;16(1):70

---

VPS35 D620N knockin mice recapitulate cardinal features of Parkinson's disease.

Niu M, Zhao F, Bondelid K, Siedlak SL, Torres S, Fujioka H, Wang W, Liu J, Zhu X
Aging cell 2021 May;20(5):e13347

---

Profound downregulation of neural transcription factor Npas4 and Nr4a family in fetal mice neurons infected with Zika virus.

Alpuche-Lazcano SP, Saliba J, Costa VV, Campolina-Silva GH, Marim FM, Ribeiro LS, Blank V, Mouland AJ, Teixeira MM, Gatignol A
PLoS neglected tropical diseases 2021 May;15(5):e0009425

---

Complement mediates neuroinflammation and cognitive decline at extended chronic time points after traumatic brain injury.

Mallah K, Couch C, Alshareef M, Borucki D, Yang X, Alawieh A, Tomlinson S
Acta neuropathologica communications 2021 Apr 20;9(1):72

---

Sensory Ganglia-Specific TNF Expression Is Associated With Persistent Nociception After Resolution of Inflammation.

Gonçalves WA, Rezende BM, de Oliveira MPE, Ribeiro LS, Fattori V, da Silva WN, Prazeres PHDM, Queiroz-Junior CM, Santana KTO, Costa WC, Beltrami VA, Costa VV, Birbrair A, Verri WA Jr, Lopes F, Cunha TM, Teixeira MM, Amaral FA, Pinho V
Frontiers in immunology 2019;10:3120

---

AAV cis-regulatory sequences are correlated with ocular toxicity.

Xiong W, Wu DM, Xue Y, Wang SK, Chung MJ, Ji X, Rana P, Zhao SR, Mai S, Cepko CL
Proceedings of the National Academy of Sciences of the United States of America 2019 Mar 19;116(12):5785-5794

---

Bilateral Implantation of Shear Stress Modifier in ApoE Knockout Mouse Induces Cognitive Impairment and Tau Abnormalities.

Nie S, Tan Y, Zhang Z, Chen G, Xiong J, Hu D, Ye K, Zhang Y, Cao X, Chen L, Zhang Z
Frontiers in aging neuroscience 2018;10:303

---

Crocin Inhibits Oxidative Stress and Pro-inflammatory Response of Microglial Cells Associated with Diabetic Retinopathy Through the Activation of PI3K/Akt Signaling Pathway.

Yang X, Huo F, Liu B, Liu J, Chen T, Li J, Zhu Z, Lv B
Journal of molecular neuroscience : MN 2017 Apr;61(4):581-589

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of IBA1 was performed by separating 50 µg of various tissue extracts by 15% SDS-PAGE. Proteins were transferred to a membrane and probed with a IBA1 Polyclonal Antibody (Product # PA5-21274) at a dilution of 1:1000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot using IBA1 Polyclonal Antibody (Product # PA5-21274). Various whole cell extracts (30 µg) were separated by 15% SDS-PAGE, and the membrane was blotted with IBA1 Polyclonal Antibody (Product # PA5-21274) diluted at 1:5,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockout of IBA1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR741364_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of IBA1 was performed by loading 30 µg of THP-1 wild type (Lane 1), THP-1 CAS9 (Lane 2), THP-1 IBA1 KO (Lane 3) whole cell extracts. The blot was probed with Anti-IBA1 Polyclonal Antibody (Product # PA5-21274) using 1:2000 dilution and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to IBA1.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot analysis of IBA1 was performed by separating 50 µg of mouse tissue extract by 15% SDS-PAGE. Proteins were transferred to a membrane and probed with a IBA1 Polyclonal Antibody (Product # PA5-21274) at a dilution of 1:1000.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot analysis of IBA1 was performed by separating 50 µg of rat tissue extract by 15% SDS-PAGE. Proteins were transferred to a membrane and probed with a IBA1 Polyclonal Antibody (Product # PA5-21274) at a dilution of 1:1,000.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-IBA1 Polyclonal Antibody (Product # PA5-21274) and a 17 kDa band corresponding to IBA1 was observed across U-937, U-937 treated with Retinoic Acid and THP-1. Whole Cell Extract-WCL (30 µg lysate) of U-937 (Lane 1), U-937 treated with Retinoic Acid (10 µM for 24 hours) (Lane 2), THP-1 (Lane 3), A-431 (Lane 4), HeLa (Lane 5) and MCF7 (Lane 6) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:2000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunocytochemistry-Immunofluorescence analysis of IBA1 was performed in THP-1 cells fixed in 4% paraformaldehyde at RT for 15 min. Green: IBA1 Polyclonal Antibody (Product # PA5-21274) diluted at 1:500. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

IBA1 Polyclonal Antibody detects Iba1 protein at cytoplasm by immunofluorescent analysis. Sample: THP-1 cells were fixed in 4% paraformaldehyde at RT for 15 min. Green: Iba1 stained by IBA1 Polyclonal Antibody (Product # PA5-21274) diluted at 1:500. Blue: Fluoroshield with DAPI .

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of IBA1 was performed using 70% confluent log phase THP-1 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with IBA1 Polyclonal Antibody (Product # PA5-21274) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766, 1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing membrane and cytoplasmic localization. Panel e represents HeLa cells having no expression of IBA1. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of IBA1 was performed using 70% confluent log phase THP-1 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with IBA1 Polyclonal Antibody (Product # PA5-21274) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766, 1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing membrane and cytoplasmic localization. Panel e represents HeLa cells having no expression of IBA1. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunocytochemistry-Immunofluorescence analysis of IBA1 was performed in THP-1 cells fixed in 4% paraformaldehyde at RT for 15 min. Green: IBA1 Polyclonal Antibody (Product # PA5-21274) diluted at 1:500. Blue: Hoechst 33342 staining.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

IBA1 Polyclonal Antibody detects Iba1 protein at cell membrane and cytoplasm by immunohistochemical analysis. Sample: Frozen-sectioned mouse brain. Green: Iba1 stained by IBA1 Polyclonal Antibody (Product # PA5-21274) diluted at 1:500. Blue: Fluoroshield with DAPI . Antigen Retrieval: ice-cold MeOH for 5 min.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

IBA1 Polyclonal Antibody detects Iba1 protein at cell membrane and cytoplasm by immunohistochemical analysis. Sample: Paraffin-embedded mouse cerebellum. Iba1 stained by IBA1 Polyclonal Antibody (Product # PA5-21274) diluted at 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

IBA1 Polyclonal Antibody detects Iba1 protein at cell membrane and cytoplasm by immunohistochemical analysis. Sample: Paraffin-embedded rat cerebellum. Iba1 stained by IBA1 Polyclonal Antibody (Product # PA5-21274) diluted at 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

IBA1 Polyclonal Antibody detects Iba1 protein on mouse fore brain by immunohistochemical analysis. Sample: Paraffin-embedded mouse fore brain. IBA1 Polyclonal Antibody (Product # PA5-21274) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

IBA1 Polyclonal Antibody detects Iba1 protein on rat hind brain by immunohistochemical analysis. Sample: Paraffin-embedded rat hind brain. IBA1 Polyclonal Antibody (Product # PA5-21274) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemical analysis of paraffin-embedded human hepatoma, using Iba1 (Product # PA5-21274) antibody at 1:500 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry (Frozen) analysis of IBA1 was performed in frozen sectioned E13.5 Rat brain tissue using IBA1 Polyclonal Antibody (Product # PA5-21274) at a dilution of 1:250 (Green). Red: beta Tubulin 3/ TUJ1, a mature neuron marker, stained by beta Tubulin 3/ TUJ1 antibody diluted at 1:500. Blue: Fluoroshield with DAPI.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry (Paraffin) analysis of IBA1 was performed in paraffin-embedded mouse brain tissue using IBA1 Polyclonal Antibody (Product # PA5-21274) at a dilution of 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry (Paraffin) analysis of IBA1 was performed in paraffin-embedded rat brain tissue using IBA1 Polyclonal Antibody (Product # PA5-21274) at a dilution of 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

IBA1 Polyclonal Antibody detects Iba1 protein at cell membrane and cytoplasm by immunohistochemical analysis. Sample: Frozen-sectioned mouse brain. Green: Iba1 stained by IBA1 Polyclonal Antibody (Product # PA5-21274) diluted at 1:500. Blue: Fluoroshield with DAPI . Antigen Retrieval: ice-cold MeOH for 5 min.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

IBA1 Polyclonal Antibody detects Iba1 protein at cell membrane and cytoplasm by immunohistochemical analysis. Sample: Paraffin-embedded mouse cerebellum. Iba1 stained by IBA1 Polyclonal Antibody (Product # PA5-21274) diluted at 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

IBA1 Polyclonal Antibody detects Iba1 protein on mouse fore brain by immunohistochemical analysis. Sample: Paraffin-embedded mouse fore brain. IBA1 Polyclonal Antibody (Product # PA5-21274) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

IBA1 Polyclonal Antibody detects Iba1 protein on rat hind brain by immunohistochemical analysis. Sample: Paraffin-embedded rat hind brain. IBA1 Polyclonal Antibody (Product # PA5-21274) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry (Frozen) analysis of IBA1 was performed in frozen sectioned E13.5 Rat brain tissue using IBA1 Polyclonal Antibody (Product # PA5-21274) at a dilution of 1:250 (Green). Red: beta Tubulin 3/ TUJ1, a mature neuron marker, stained by beta Tubulin 3/ TUJ1 antibody diluted at 1:500. Blue: Fluoroshield with DAPI.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry (Paraffin) analysis of IBA1 was performed in paraffin-embedded mouse brain tissue using IBA1 Polyclonal Antibody (Product # PA5-21274) at a dilution of 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow Cytometry analysis of S100A7 was performed in THP-1 cells using S100A7 Monoclonal Antibody (47C1068) (Product # MA1-91555) (red) at a dilution of 1:50. Black: Unlabelled sample was used as a control. Acquisition of 20,000 events were collected using a Dylight 488-conjugated secondary antibody for FACS analysis.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow Cytometry analysis of S100A7 was performed in THP-1 cells using S100A7 Monoclonal Antibody (47C1068) (Product # MA1-91555) (red) at a dilution of 1:50. Black: Unlabelled sample was used as a control. Acquisition of 20,000 events were collected using a Dylight 488-conjugated secondary antibody for FACS analysis.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig 3. Complement C3 deposition decreases with continuous CR2Crry treatment. a Representative Imaris images of processed 40x z-stacked immunofluorescence images acquired by confocal microscopy from two regions adjacent to site of TBI impact (as shown in the atlas image represented in this figure) from vehicle treatment, 1-week CR2Crry treatment, continuous CR2Crry treatment, and naive mice (9 months). Hoechst (blue), C3 (red), NeuN (green), and Iba-1 (aqua). b Quantification of C3 intensity. c Quantification of number of C3/NeuN interactions per frame. d Quantification of number of C3/NeuN/Iba1 interaction per frame. One-way ANOVA with Bonferroni correction for multiple comparisons. Unpaired T-test: continuous vs. vehicle. Error bars = Mean +/- SEM. * p < 0.05, ** p < 0.01. n = 6-9/group. e 3D representative example of interactions (white arrows) between C3 (red)/ NeuN (green) and C3 (red)/ NeuN (green)/ Iba1 (aqua).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig 4. Continuous CR2Crry treatment decreases area of microgliosis in the ipsilateral hemisphere. a 4x stitched immunofluorescence images of Iba-1 (red) microglia/macrophage staining in the three conditions of the study: vehicle, 1-week CR2Crry, and continuous CR2Crry from two different brain coordinates: Bregma - 0.18 mm (+/-0.04) and Bregma - 1.46 mm (+/-0.04). b Quantification of stained images. One-way ANOVA with Bonferroni correction for multiple comparisons. Unpaired T-test: continuous vs. vehicle. Error bars = Mean +/- SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. n = 8-11/group.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig 6. CR2Crry treatment decreases astrocytosis in the contralateral hippocampus. a Representative images of contralateral hippocampus Iba-1 staining for each of the following conditions: vehicle, 1-week CR2Crry, continuous CR2Crry, and naive 9 month old mice. Quantification of Iba-1 intensity (corrected tissue fluorescence) is also shown. One-way ANOVA with Bonferroni correction for multiple comparisons. Unpaired T-test: continuous versus vehicle. Error bars = Mean +/- SEM. ** p < 0.01. Vehicle, 1-week, continuous (n = 6-8) and naive (n = 3). b Representative images of contralateral hippocampus GFAP staining for each of the following conditions: vehicle, 1-week CR2Crry, continuous CR2Crry, and naive 9 month old mice. Quantification of GFAP intensity (corrected tissue fluorescence) is also shown. One-way ANOVA with Bonferroni correction for multiple comparisons. Unpaired T-test: continuous vs. vehicle. Error bars = Mean +/- SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001. Vehicle, 1-week, continuous (n = 6-8) and naive (n = 3).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

FIGURE 6 Increased MPTP vulnerability of VPS35 D620N KI mice. (a) A schematic diagram depicts the experimental design. (b-c) Exacerbated motor function deficits in VPS35 D620N / D620N mice as measured by the latency to fall in rotarod test (b) and duration to cross the narrow beam (9 mm) in beam walking test (c) ( n = 12-14/group). (d-g) Concentrations of DA (d), DOPAC (e), HVA (f), and DA turnover rate (DOPAC+HVA/DA) (g) in the STR of VPS35 D620N / D620N and WT controls injected with MPTP/Saline as indicated ( n = 10-12/group). (h) Representative images of TH, GFAP, and IBA1 immunostaining in SNpc and STR from VPS35 D620N / D620N and WT mice injected with MPTP/Saline as indicated. Scale bar, 100 mum. Quantification of TH-positive neurons in SNpc (i), OD of TH-positive fibers in STR (j), GFAP-positive cells (k) and IBA1-positive cells (l,m) in SNpc ( n = 9/group). Representative Western blots (n) and quantification (o) of TH in VM extracts from VPS35 D620N / D620N mice and WT controls injected with MPTP/Saline as indicated ( n = 7/group). Two-way ANOVA with Tukey's post hoc test; mean +- SEM ; * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., not significant

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

FIGURE 2 Neuropathological changes in VPS35 D620N KI mice. (a,e) Representative images of TH, GFAP and IBA1 immunostaining in SNpc (a) and STR (e) of 15- to 16-month-old VPS35 D620N / D620N and WT mice ( n = 6-9/group). Scale bar, 100 mum. Quantification of TH-positive neurons (b), GFAP-positive cells (c) and IBA1-positive cells (d) in SNpc. Quantification of optical density (OD) of TH-positive fibers (f), GFAP-positive cells (g), and IBA1-positive cells (h) in STR. (i-k) Representative Western blots (i) and quantification of TH (j) and GFAP (k) in VM and STR extracts from 15- to 16-month-old VPS35 D620N / D620N mice and WT controls ( n = 7/group). Student's t test, unpaired, two-tailed; data are shown as mean +- SEM ; * p < 0.05; ** p < 0.01; n.s., not significant

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig. 3 Decreased neuronal METTL3 and m 6 A in the hippocampus by AAV-shMettl3. (A) Highly conserved gene sequences of shRNA targeting region in mouse and rat Mettl3 mRNA. (B) Knockdown effect of shMettl3 was confirmed in mouse N2a cells 3 days after transient transfection (Data are means+-SEM from 3 independent experiments). (C) AAV-mediated gene delivery in the hippocampus was confirmed by immunostaining for GFP. WT C57BL/6 mice receiving bilaterally stereotaxic injections of AAV encoding for eGFP plus anti-Mettl3 shRNA (shMettl3) or scrambled control shRNA (shCtrl) into the hippocampus at 2 months of age were analyzed 1 month later. (D-F) Representative images of the AAV-infected areas (GFP-positive) in hippocampus for different cell markers by immunocytochemistry after fixation: MAP 2 for neurons (D), GFAP for astrocytes (E), and Iba1 for microglia (F). (G, H) Representative image of immunoreactivity of METTL3 or m 6 A (G) in AAV-shRNA-injected mice and quantification analysis (H) confirmed that METTL3 protein and m 6 A modification level were reduced in mice injected with shMettl3 virus (n = 5-7 mice per group). GFP staining has been performed in adjacent brain slices at the same time and immunoreactivities of m 6 A and METTL3 were measured in GFP-positive regions. (Data are means+-SEM, * p < 0.05, **p < 0.01, (B, H) unpaired student's t-test)

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Additional file 2: Supplementary Fig. 2. m6A is increased in astrocytes in AD hippocampus, but limited colocalization of m6A with Iba1 in AD was observed. (A-B) Colocalization of m6A (Novus, A; SYSY, B) with astrocyte marker GFAP (Thermofisher, A; MP Biomedicals, B) in hippocampal tissues from AD and control brains. (C) Quantification revealed that m6A immunoreactivity was increased in astrocytes in AD hippocampal tissues compared with control. (D) AD and control hippocampal sections were stained for m6A (Novus) and Iba1. Only some colocalization of m6A and Iba1 was observed in AD and control hippocampal sections. (E) Negative control experiments were performed without primary antibody in immunostaining for m6A modification in human brain cases (n = 5-6 in each group, *p < 0.5, C, unpaired student's t-test).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Additional file 3: Supplementary Fig. 3. Validation of intracranial injection into hippocampus by needle track and induced neuroinflammation by METTL3 depletion in mouse hippocampus. (A) A representative image of needle track (arrow) of AAV-injected mice was shown. GFP immunoreactivity was detected in area adjacent to the needle tack. NeuN staining revealed severe neuronal loss around injected areas only in AAV-shMettl3 injected mice but not AAV-shCtrl injected mice. (B-E) Representative images of immunohistochemistry for Iba1 (B) and GFAP (Thermofisher, D) in hippocampal CA1/2 or CA3 areas in shRNA-injected mice and their quantification (C for astrocyte and E for microglia) analysis showed that METTL3 knockdown caused neuroinflammation in mouse hippocampus. (n = 4-7, *p < 0.5, **p < 0.01; C, E, unpaired student's t-test).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 2. ZIKV exposure during the embryonic period does not lead to an inflammation that persists until adulthood. Shown are representative images for S100-beta immunostaining of the hippocampal CA1 region from MOCK female (A), ZIKV female (B), MOCK male (D), and ZIKV male (E) mice. Scale bar = 50 mum. Graphs show quantification of S100-beta-labeled hippocampal CA1 astrocytes per 62500 mum 2 in MOCK and ZIKV-exposed female (C) and male (F) mice. Data represent the means +- SEM obtained from 3 images taken from 3 histological slices per mouse. Shown are representative images for IBA-1 immunostaining of the hippocampal CA1 region from MOCK female (G), ZIKV female (H), MOCK male (J), and ZIKV male (K) mice. Scale bar = 50 mum. Graphs show quantification of IBA-1-labeled hippocampal CA1 microglia per 62500 mum 2 in MOCK and ZIKV-exposed female (I) and male (L) mice. Data represent the means +- SEM obtained from 3 images taken from 3 histological slices per mouse. Graphs show mRNA levels of IL-1beta (M, Q), TNF-alpha (N, R), IL-6 (O, S) and CCL2 (P, T) in hippocampal samples from MOCK and ZIKV-exposed female and male mice. mRNA levels were assessed by quantitative RT-PCR, which was performed in duplicate and normalized to RPL32 mRNA levels.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 4 Resident macrophage activation and inflammatory molecules gene expression characterizes inflammatory status of DRG after resolution of inflammation in AIA model. Immunized mice received either i.a. injection of mBSA (10 mug/10 mul of PBS) to AIA induction or PBS (10 mul) in control group. (A) Confocal microscopy slides show marked Iba1 + cells within DRG (L4) 8 days after AIA induction and (B) its quantification ( n = 5). Time course of gene expression of (C) IL-1beta, (D) IL-6, (E) CXCL1, (F) COX-2, (G) iNOS, and (H) IL-10 within lumbar DRG (L3-L5). DRG samples from the control group were extracted 8 days after i.a. injection ( n = 5). The results are represented as mean +- SEM; p = specified value in the graph compared to control group (PBS) using ANOVA following by Dunnett post-hoc analysis.