All stainings were performed on 10 μm fresh-frozen sections. Oil red O (ORO) staining as well as hematoxylin and eosin (HE) staining of liver sections was performed to define liver histopathologies. Immunoflorescent (IF) stainings of mouse liver sections were performed using the following primary antibodies: anti-mouse/human ApoE (ab52607; Abcam); anti-mouse C1q (ab182451; Abcam); anti-mouse CLEC4F (ab2608299; Invitrogen); anti-mouse CD68 (FA11; Serotec); anti-mouse CD31 (ab553370; BD PharMingen); anti-human C3 (A213; ComplementTech); anti-mouse C4 (HM1046; Hycult Biotech); anti-mouse C5 (ab11898, Abcam). We had previously established the specificity of IF microscopy in mouse and human tissues including no primary antibody negative controls, isotype antibody controls, ApoE knockout mouse tissue (i.e., anti-ApoE antibodies) (1 (link)). IF stainings of human sections were performed using the following antibodies: anti-mouse/human ApoE (ab52607; Abcam); anti-human C1q (ab71089; Abcam); anti-human CD68 (EMB11; DAKO); anti-human C5 (A220; ComplementTech). Hepatitis sections as well as the appropriate control tissues were fixated with Delaunay solution prior to IF staining. Stained sections were analyzed using a Leica confocal microscope (SP8, Leica, Germany) using Leica Application Suite (Leica) and ImageJ software.
Ab182451
Manufactured by Abcam
Ab182451 is a laboratory product designed for research purposes. It serves as a tool for scientific investigations, but its specific core function is not detailed here to maintain an unbiased and factual approach.
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Lab products found in correlation
Lsm 800, by Zeiss (2 mentions)
Alexa fluor 647plus, by Thermo Fisher Scientific (2 mentions)
Vectastain elite abc hrp detection kit, by Vector Laboratories (2 mentions)
Mca1957, by Abcam (2 mentions)
Ba 1000, by Vector Laboratories (2 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (2 mentions)
Confocal microscope, by Leica (1 mentions)
Application suite, by Leica (1 mentions)
Ab52607, by Abcam (1 mentions)
Ab71089, by Abcam (1 mentions)
Ab2251, by Abcam (1 mentions)
Nf κb p65 d14e12 xp rabbit mab, by Cell Signaling Technology (1 mentions)
Neuronal nuclei (neun), by Synaptic Systems (1 mentions)
Ab53554, by Abcam (1 mentions)
Ab6142, by Abcam (1 mentions)
Ab13970, by Synaptic Systems (1 mentions)
Camkii α 6g9 mouse monoclonal antibody mab, by Cell Signaling Technology (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Dm5500 microscope, by Leica (1 mentions)
11 protocols using ab182451
1
Comprehensive Liver Histopathology and Immunofluorescence Analysis
2
Synaptic Staining of Brain Sections
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3
Immunohistochemistry for Neural Markers
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The following antibodies were used: anti–phospho-histone H2A.X (Ser139) antibody, clone JBW301, EMD Millipore, 05-636, 1:500; NeuN, Synaptic Systems, 266 004, 1:1000; CaMKII-α (6G9) mouse monoclonal antibody (mAb), Cell Signaling Technology, 50049S, 1:200; NEUROD1 polyclonal antibody, Proteintech, 12081-1-AP, 1:200; anti-GFP antibody, Abcam, ab13970, 1:500; Iba1, Synaptic Systems, 234 004, 1:1000; anti-GFAP antibody, Abcam, ab53554, 1:500; recombinant anti-Olig2 antibody (EPR2673), Abcam, ab109186, 1:500; RFP antibody preadsorbed, Fisher Scientific, 600-401-379, 1:200; NFκB p65 (D14E12) XP rabbit mAb, Cell Signaling Technology, 8242S, 1:500; NFκB p65 polyclonal antibody, Invitrogen, 51-0500, 1:100; anti–MHC class II (I-A/I-E), clone M5/114, EMD Millipore, MABF33, 1:500; anti-nestin antibody (rat 401), Abcam, ab6142, 1:1000; anti-C1q antibody (4.8), Abcam, ab182451, 1:500; synapsin 2 antibody (guinea pig), Synaptic Systems, 106 004, 1:500; VGlut1 (rabbit), Synaptic Systems, 135 303, 1:500; purified anti-mouse/rat β-amyloid antibody, BioLegend, 805801, 1:500; phospho-Tau (Thr181) (D9F4G) rabbit mAb, Cell Signaling Technology, 12885S, 1:500.
4
Multiplex Immunohistochemistry for Brain Samples
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Brain samples were coronally cut into 25‐μm‐thick slices. Multiplex immunohistochemistry (mIHC) was performed by Opal Polaris 7‐Color Manual IHC Detection Kit (Akoya Biosciences, NEL861001KT), following the manufacturer's instructions. The following primary antibodies were used: anti‐CD8α (Abcam, ab217344, 1:100), anti‐C1Q (Abcam, ab182451, 1:100), and anti‐APOE (Abcam, ab183596, 1:100). Sections were observed with the automated imaging system Vectra® 3.0 (Akoya Biosciences).
Immunofluorescence staining was performed as previously described.
2 (link) Briefly, after blockage with 5% BSA in 0.3% triton X‐100 for 1 h at room temperature, the brain sections were incubated overnight at 4°C with the following primary antibodies: anti‐GPNMB (Abcam, ab188222, 1:250), anti‐MHCII (Thermo Fisher, 14–5321‐85, 1:100), and anti‐IBA1 (Abcam, ab5076, 1:200). Then the sections were incubated with the following secondary antibodies: donkey anti‐rabbit Alexa Fluor 647, (Invitrogen, 1:500), donkey anti‐rat Alexa Fluor 488, (Invitrogen, 1:500), donkey anti‐goat Alexa Fluor 555 (Invitrogen, 1:500). A Leica DM5500 microscope was used to observe the sections and capture images.
Immunofluorescence staining was performed as previously described.
2 (link) Briefly, after blockage with 5% BSA in 0.3% triton X‐100 for 1 h at room temperature, the brain sections were incubated overnight at 4°C with the following primary antibodies: anti‐GPNMB (Abcam, ab188222, 1:250), anti‐MHCII (Thermo Fisher, 14–5321‐85, 1:100), and anti‐IBA1 (Abcam, ab5076, 1:200). Then the sections were incubated with the following secondary antibodies: donkey anti‐rabbit Alexa Fluor 647, (Invitrogen, 1:500), donkey anti‐rat Alexa Fluor 488, (Invitrogen, 1:500), donkey anti‐goat Alexa Fluor 555 (Invitrogen, 1:500). A Leica DM5500 microscope was used to observe the sections and capture images.
5
Immunohistochemistry of Brain Tissue
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Tissue processing and immunohistochemistry was performed on free-floating sections according to standard published techniques26 (link). Cryoprotected brains were sectioned coronally at 40 μm with a cryomicrotome (Leica Camera). Free-floating coronal sections (40 μm) were incubated overnight with anti-IBA1 (1:1,000, Wako, 0191741; or 1:1,000, Synaptic Systems, 234-004), rat anti-CD68 (FA-11, 1:250, Bio-Rad MCA1957), rabbit anti-C1q (1:500, Abcam ab182451) and anti-phosphorylated-CREB (Ser133) (1:2,500, Millipore 06-519) primary antibodies. Labelling was revealed using secondary antibodies (donkey anti-rabbit conjugated Alexa Fluor 555 (1:750, Life Technologies, A31572), donkey anti-rat conjugated Alexa Fluor 647PLUS (1:750, Invitrogen, A48272) donkey anti-guinea pig conjugated Alexa Fluor 488 (1:750, Jackson ImmunoResearch, 706-545-148) and goat anti-rabbit, biotinylated (1:500, Vector, BA-1000)). Labelling of biotinylated antibodies was revealed using the Vectastain Elite ABC-HRP Detection Kit (Vector, PK-6100) with diaminobenzidine (Sigma-Aldrich, D5905). Sections were imaged using confocal microscopy (Zeiss LSM800 or Zeiss LSM900) or bright-field microscopy (Keyance). Individual cell numbers and intensity in the dentate gyrus was quantified across 3–4 sections per animal using ImageJ.
6
Immunohistochemical Analysis of Neuroinflammation
7
Quantifying Microglial C1q Expression
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The immortalized murine microglial BV-2 cell line was grown and maintained in Dulbecco’s modified Eagle serum (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in 5% CO2. BV-2 cells were collected, and 1 × 105 cells were plated in 6-well plates overnight. The medium was removed, and cells were stimulated with either vehicle (1% ethanol) or 10nM Ro5 (Sigma Aldrich, C5174) diluted in DMEM-Ham’s F12 containing 1% penicillin-streptomycin for 24 h, followed by stimulation with 100ng/ml LPS or vehicle for 2h. Cells were then trypsinized and centrifuged, and the resulting pellet was fixed in 4% paraformaldehyde for 10 min, followed by permeabilization with 0.1% Triton X-100 in phosphate-buffered saline for 5 min. C1q was labeled using an anti-C1q antibody (Abcam, ab182451), and fluorochrome-conjugated anti-rabbit secondary antibody (Invitrogen, A11034), and fluorescence was detected by flow cytometry. Mean fluorescent intensity was analyzed using the FlowJo software (Tree Star, Ashland, OR).
8
Quantifying C1q Immunoreactivity in Gustatory Projections
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An antibody to C1q (rabbit; 1:1,000; Cat# ab182451, RRID:AB_2732849; Abcam) was used to examine the overlap of staining in P25 mice. Standard perfusion and fixation procedures were followed and described above. Elements software (v.5.02; Nikon) was used to automatically draw a region of interest around the densest P2X2+ label (gustatory projection) and draw a region of interest around all the P2X2+ label. These two regions of interest were then used to calculate the density of C1q+ label within each of the two regions of interest. From these measures, the densities within the entire P2X2+ field, within the core of the P2X2+ field, and of the C1q+ label between the core and outermost region of interest (i.e., around the fringe of the terminal field) were determined.
9
Immunohistochemical Analysis of Tau Pathology and Neuroinflammation
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Coronal or sagittal sections (35 µm thickness) were washed with 0.1% Triton in PBS, saturated by incubation with 0.1% Triton in PBS/5% goat serum, and then incubated with primary antibodies as follows: AT8 anti-P-tau pSer202/Thr205 (1/500, Thermo Scientific, #MN1020); anti-P-tau pS396 (1/1000, Abcam, #ab109390); anti-IBA1 (1/1000, Wako, #019-19741), CD68 (1/500, Bio-Rad, #MCA1957) and anti-C1q (1/1000, Abcam, #ab182451). For fluorescent immunostaining, sections were incubated with the appropriate secondary antibody: anti-rabbit IgG Alexa Fluor 488 or 568 (1/2000, Invitrogen); anti-mouse IgG Alexa Fluor 488 or 568 (1/2000, Invitrogen). For non-fluorescent immunostaining, endogenous peroxidase was quenched with PBS containing 3% H2O2 for 15 min followed by amplification using the ABC system (VECTASTAIN Elite ABC HRP Kit, Vector Laboratories, Burlingame, CA, USA). Horseradish peroxidase conjugates and 3,3′-diaminobenzidine were used according to the manufacturer’s manual (Vector® DAB, Vector Laboratories, Burlingame, CA, USA). Images were obtained with an Olympus BX61 microscope and analyzed with Fiji software (ImageJ).
10
Immunohistochemistry of Brain Tissue
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Tissue processing and immunohistochemistry was performed on free-floating sections according to standard published techniques26 (link). Cryoprotected brains were sectioned coronally at 40 μm with a cryomicrotome (Leica Camera). Free-floating coronal sections (40 μm) were incubated overnight with anti-IBA1 (1:1,000, Wako, 0191741; or 1:1,000, Synaptic Systems, 234-004), rat anti-CD68 (FA-11, 1:250, Bio-Rad MCA1957), rabbit anti-C1q (1:500, Abcam ab182451) and anti-phosphorylated-CREB (Ser133) (1:2,500, Millipore 06-519) primary antibodies. Labelling was revealed using secondary antibodies (donkey anti-rabbit conjugated Alexa Fluor 555 (1:750, Life Technologies, A31572), donkey anti-rat conjugated Alexa Fluor 647PLUS (1:750, Invitrogen, A48272) donkey anti-guinea pig conjugated Alexa Fluor 488 (1:750, Jackson ImmunoResearch, 706-545-148) and goat anti-rabbit, biotinylated (1:500, Vector, BA-1000)). Labelling of biotinylated antibodies was revealed using the Vectastain Elite ABC-HRP Detection Kit (Vector, PK-6100) with diaminobenzidine (Sigma-Aldrich, D5905). Sections were imaged using confocal microscopy (Zeiss LSM800 or Zeiss LSM900) or bright-field microscopy (Keyance). Individual cell numbers and intensity in the dentate gyrus was quantified across 3–4 sections per animal using ImageJ.
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