rat  (Miltenyi Biotec)

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: Infiltration of CD68-positive activated microglia/macrophages co-expressing PSAP and PGRN into the SFO and surrounding tissues a – b) Double immunofluorescent staining of PGRN (green) and CD68 (red) around the SFO in 10-month-old-female WT and SAP-D −/− mice. b , Magnified images of the indicated white squares in a . ⅰ: SFO, ⅱ: Fornix, and ⅲ: Perivascular, bv: blood vessel. c ) Quantification of PGRN- and/or CD68-staining in the SFO and surrounding areas in WT and SAP-D −/− mice. Data are shown as the mean ± SD (n = 3). The left panel presents a stacked bar chart, whereas the right panel shows the individual data values in a bar chart format. d – h ) Triple immunofluorescent staining of PSAP (red), PGRN (green), and CD68 (cyan) around the SFO in 10-month-old-female SAP-D −/− mice. e) Magnified images of the indicated white squares in d) ⅳ: Boundary, ⅴ: Fornix, and ⅵ: Perivascular. White arrowheads indicate triple co-staining with PSAP, PGRN, and CD68. Open arrowheads indicate PGRN signals alone. Co-localization rates of CD68 positive areas in PSAP ( f ), PGRN ( g ), and PSAP-PGRN staining areas ( h ) around the SFO of SAP-D −/− mice, respectively. f-h ) The left panel presents a stacked bar chart, whereas the right panel presents the individual data values in a bar chart format. Data are shown as mean ± SD (n = 3). Nuclei are labeled by DAPI (blue) staining. All scale bars, 50 μm.

Article Snippet: The primary antibodies used were a rabbit antibody against PSAP (dilution 1:100, 10801-1-AP, Proteintech Group Inc., IL, USA), a sheep antibody against PGRN (dilution 1:100, AF 2557, R&D Systems Inc., MN, USA), a guinea pig antibody against c-Fos (dilution 1:500, 226308, Synaptic Systems GmbH, Göttingen, Germany), a rat antibody against CD68-FITC conjugated (dilution 1:500, MCA1957FA, BIO-RAD Laboratories Inc., CA, USA), and a rat antibody against LAMP1 (dilution 1:100, ab25245, Abcam, Cambridge, UK).

Techniques: Expressing, Staining, Labeling

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: c-Fos expression was induced in the SFO of SAP-D −/− mice without dehydration. a) Expression levels of c-Fos , Gpr37 , and Cd68 in the SFO and fornix were quantified via RT-qPCR. For RNA extraction, tissue samples were microdissected from the brains of 6-month-old female WT mice (n = 10) and SAP-D −/− mice (n = 6) under a stereomicroscope. After cDNA synthesis, RT-qPCR was performed, and the data were normalized to Gapdh expression. A significant increase in Cd68 expression was observed in SAP-D −/− mice, consistent with immunostaining results, confirming accurate sampling of the SFO and surrounding fornix. The results of Student's t-tests for each panel are as follows: left panel, p = 0.0046, Cohen's d = 1.74 (95 % CI: 0.0027, 0.0122); center panel, p = 0.0352, Cohen's d = 1.20 (95 % CI: 0.00091, 0.02191); and right panel, p = 0.0007, Cohen's d = 2.41 (95 % CI: 0.0079, 0.0233). b) Immunofluorescent staining of c-Fos (green) in the SFO of 10-month-old female WT and SAP-D −/− mice. WT and SAP-D −/− mice had free access to drinking water (indicated as FD) or 24 h water deprivation (indicated as DH). The white dotted lines enclose the SFO. Nuclei are labeled by DAPI (blue) staining. Scale bars, 50 μm. c) Percentage of c-Fos positive cells among all DAPI stained cells in the SFO (%). Data are shown as the mean ± SD (n = 7). , , , and indicate the individual values in each group. Two-way ANOVA showed significant main effects of water deprivation, F(1,24) = 35.13, p < 0.0001, ηp 2 = 0.13, 95 % CI (−8.67, −4.19), and genotype, F(1,24) = 130.40, p < 0.0001, ηp 2 = 0.36, 95 % CI (−14.64, −10.16), as well as a significant water deprivation × genotype interaction, F(1,24) = 38.60, p < 0.0001, ηp 2 = 0.14, 95 % CI (−17.98, −9.01). Post-hoc Tukey's tests showed significant differences between WT-FD versus WT-DH ( p < 0.0001, Cohen's d = 5.83 [95 % CI: −17.42, −8.94]), WT-FD versus SAP-D −/− -FD ( p < 0.0001, Cohen's d = 6.91 [95 % CI: −23.38, −14.91]), and WT-DH versus SAP-D −/− -DH ( p = 0.006, Cohen's d = 1.90 [95 % CI: −9.89, −1.41]), but no significant difference between SAP-D −/− -FD versus SAP-D −/− -DH ( p = 0.997, Cohen's d = 0.09 [95 % CI: −3.92, 4.54]). ns: no significant difference. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: The primary antibodies used were a rabbit antibody against PSAP (dilution 1:100, 10801-1-AP, Proteintech Group Inc., IL, USA), a sheep antibody against PGRN (dilution 1:100, AF 2557, R&D Systems Inc., MN, USA), a guinea pig antibody against c-Fos (dilution 1:500, 226308, Synaptic Systems GmbH, Göttingen, Germany), a rat antibody against CD68-FITC conjugated (dilution 1:500, MCA1957FA, BIO-RAD Laboratories Inc., CA, USA), and a rat antibody against LAMP1 (dilution 1:100, ab25245, Abcam, Cambridge, UK).

Techniques: Expressing, Quantitative RT-PCR, RNA Extraction, cDNA Synthesis, Immunostaining, Sampling, Staining, Labeling

Journal: Nature Communications

Article Title: A cholesterol-responsive hepatic tRNA-derived small RNA regulates cholesterol homeostasis and atherosclerosis development

doi: 10.1038/s41467-025-67387-z

Figure Lengend Snippet: Seven-week-old male Ldl receptor -deficient ( Ldlr -/- ) mice were treated with tsRNA-Glu-CTC antisense oligonucleotides (ASO) or control ASO twice per week for 7 weeks. The mice were maintained on a normal chow diet (ND) or switched to a high-cholesterol diet (HCD) starting at 8-week-old age. a The schematic of tsRNA-Glu-CTC ASO treatment experiment. Created in BioRender. Zhou, C. ( https://BioRender.com/rj9jwzr ). b – g Body weight ( n = 8,10,10) ( b ), representative photos of collected serum ( c ), serum total cholesterol ( n = 8,10,10) (left panel) and triglyceride ( n = 5,10,10) (right panel) levels ( d ), cholesterol levels of lipoprotein fractions (VLDL-C, LDL-C, and HDL-C) ( n = 8,10,10) ( e ), representative Oil-Red-O (top) and hematoxylin and eosin (bottom) stained liver sections (scale bar =100 μm) ( f ), and hepatic cholesterol ( n = 6,9,6) (left panel) and triglyceride ( n = 6,8,6) (right panel) contents ( g ) of Ldlr -/- mice treated with vehicle control, control ASO, or tsRNA-Glu-CTC ASO (data are shown as mean ± SEM, one-way ANOVA, Bonferroni multiple-comparison test). h Representative images of Oil-red-O-stained sections and quantitative analysis of the atherosclerotic lesion area at the aortic root of Ldlr -/- mice ( n = 7,8,8; mean ± SEM, one-way ANOVA, Bonferroni multiple-comparison test). i Correlation between serum total cholesterol levels and atherosclerotic lesion sizes in Ldlr -/- mice ( n = 16; Correlation was analyzed by Pearson correlation). j Representative images of immunofluorescence staining of CD68 (green) and α-smooth muscle actin (αSMA) (red) at the aortic root of Ldlr -/- mice (scale bar=200 µm). Quantitative analysis of staining area is displayed as indicated ( n = 6,6,6; mean ± SEM, one-way ANOVA, Bonferroni multiple-comparison test). k The expression levels of hepatic lipogenic genes of HCD-fed Ldlr -/- mice treated with tsRNA-Glu-CTC ASO or control ASO were analyzed by quantitative real-time PCR ( n = 6, mean ± SEM, two-tailed Student’s t-test). n represents the number of biological replicates (mice). VLDL-C very low-density lipoprotein cholesterol; LDL-C low density lipoprotein cholesterol; HDL-C high density lipoprotein cholesterol.

Article Snippet: For the immunofluorescence staining, samples were fixed in 4% PFA for 15 min and permeabilized with 0.5% Triton X-100 in PBS for 15 min. After incubating with 5% BSA for 1 hr at room temperature, the slides were incubated with primary antibodies against CD68 (Bio-Rad, MCA1957, 1:100) and α-SMA (Abcam, ab5694, 1:100) at 4 °C for 12 to 16 hr , .

Techniques: Control, Staining, Comparison, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Brain-infiltrated monocyte macrophages in a rat model of temporal lobe epilepsy: revisiting the pro-inflammatory paradigm

doi: 10.3389/fimmu.2025.1695856

Figure Lengend Snippet: Peripheral monocytes infiltrate the hippocampus following SE between +7 h and +6 days and differentiate into brain monocyte-macrophages. (A-D) Fluorescent YG carboxylate microspheres (FYG, 0.5 µm) were injected into the tail vein 6 h after SE. No labeled monocytes could be detected in the brain parenchyma unless circulating monocytes were depleted with clodronate liposomes (1 ml/100g; i.p.) administered prior to SE. The rats were sacrificed 1D, 3D and 6D after SE. Detection of CD11b (red, CBL1512Z, Millipore) and FYG (green) in infiltrating monocytes at I day [ (B) cap—capillary], brain monocyte-macrophages with extending processes at 3 days post-SE (C) and in cells resembling activated microglia at 6 days post-SE (D) in the hilus. Scale: 20 µm. (E–N) CD11b (E— l, cyan, CBL1512Z, Millipore) and CD68 [ (J–N) green, MCA341GA, Bio-Rad] were immunodetected in the dentate gyrus following SE (CTRL, n=6; SE+7 h, n=4; SE+ID, n=5; SE+6D, n=4). Scale: 50 µm. Round CD11b-positive cells (J) and CD68-positive cells (N) were quantified in the dentate gyrus. The data were analyzed with Tukey's test following one-way ANOVA. The data are presented as the means ± SEMs. vs. CTRL. p<0.0001.

Article Snippet: Infiltrating monocytes and monocyte-macrophages (mo-MΦ) were labelled with mouse anti-CD68 antibody (1:1000, MCA341GA, Bio-Rad).

Techniques: Injection, Labeling, Liposomes

Journal: Frontiers in Immunology

Article Title: Brain-infiltrated monocyte macrophages in a rat model of temporal lobe epilepsy: revisiting the pro-inflammatory paradigm

doi: 10.3389/fimmu.2025.1695856

Figure Lengend Snippet: Infiltrating monocytes differentiate into brain monocyte-macrophages bearing morphological feature of microglia. (A–J) . CD68 (MCA341GA, Bio-Rad) and Iba1 (ab5076, abcam) were immunodetected in the dentate gyrus (left) and CA1 region (right), in control rats and 7 h, 1D, 9D and 7 W post-SE. Images were acquired with a slide scanner, objective ×20. Scale bars: 20 µm. (K, L) . Quantification of round and ramified CD68-positive cells in the hilus and in CA1 region, CTRL, n=5; SE+7 h, n=4; SE+1D, n=5; SE+9D, n=4; SE+7 W, n=5. Data are analyzed with Tukey’s test following two-way RM ANOVA. (M, N) . Percentage of Iba1-positive cells among CD68-positive cells was quantified in the hilus (M) and in CA1 region (N), CTRL, n=3; SE+7 h, n=3; SE+1D, n=4; SE+9D, n=4; SE+7 W, n=3. Only the time points at which a sufficient number of CD68-positive cells were detected (>20 cells/mm2), allowing the percentage of Iba1 + CD68 + cells to be meaningfully interpreted, i.e., from 1 day post-SE onward, were included in Figure (M, N) . Data are analyzed with Tukey’s test following One-way ANOVA (M) or with Dunn’s test following Kruskall-Wallis test (N). *: vs. SE+1D. Data are presented as the mean + SEM. *p<0.05; ***p<0.001; ****p<0.0001. Only significant differences between round and ramified cells are shown in (K, L) . All statistical tests are detailed in .

Article Snippet: Infiltrating monocytes and monocyte-macrophages (mo-MΦ) were labelled with mouse anti-CD68 antibody (1:1000, MCA341GA, Bio-Rad).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Brain-infiltrated monocyte macrophages in a rat model of temporal lobe epilepsy: revisiting the pro-inflammatory paradigm

doi: 10.3389/fimmu.2025.1695856

Figure Lengend Snippet: Proposed time course of entry and integration of monocytes infiltrating inflamed brain regions after pilocarpine-induced SE in rats. The blood—brain barrier (BBB) state following SE is depicted in the boxes at the top of the diagram . Myeloid cells in the hippocampus, the VLR and the ThD parenchyma, i.e., microglia and monocytes, are represented in the boxes at the bottom. Markers enabling the staining of both cell types at the histological level (but not at the transcript level) are highlighted in green for microglia and purple for monocytes/monocyte-macrophages (mo-mΦs). At 7 hours after SE, microglia are primarily responsible for the extensive expression of proinflammatory cytokines, and monocytes have not yet invaded brain tissue. By 24 hours after SE, while inflammation remains at a significant level, a substantial number of round monocytes are observed, contributing to the expression of IL-1ß but displaying a predominantly neuroprotective phenotype. Nine days after SE, a glial scar composed mainly of microglia and brain mo-mΦs formed, although the inflammatory response was largely resolved. At this stage, some mo-mΦs start to coexpress the monocyte marker CD68 and the microglia marker Iba1. During the chronic epilepsy phase, microglia return to a basal inflammatory state, but low-grade inflammation persists, likely due to the presence of mo-mΦs. BBB, blood—brain barrier; CTRL, control, resting condition; D, day; SE, status epilepticus; W, week.

Article Snippet: Infiltrating monocytes and monocyte-macrophages (mo-MΦ) were labelled with mouse anti-CD68 antibody (1:1000, MCA341GA, Bio-Rad).

Techniques: Staining, Expressing, Marker, Control