sheep antibody against pgrn  (R&D Systems)

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: Increased protein levels of PSAP and PGRN within the SFO of SAP-D −/− mice a – b) , Coronal brain section at 0.7–0.8 mm posterior to bregma containing the SFO. a: DAPI staining. a-2: Enlarged view of the white square in a-1. SFO: subfornical organ, 3V: third ventricle. b; Double immunofluorescent staining of PSAP (red) and PGRN (green) in 10-month-old female WT and SAP-D −/− mice. DAPI (blue) staining showed the nuclei ( a and b ). Scale bar, 500 μm ( a and b ). White arrowheads indicate the SFO regions ( b ). c) Cerebral region from 3-, 6-, and 10-month-old male and female mice containing the SFO (0.7–0.8 mm posterior to ∗bregma) used for protein extraction and Western blot ( d – h ) using anti- PSAP, PGRN, and GAPDH antibodies. Quantification normalized to GAPDH expression and represented as the mean ± SD of three mice for each group. and indicate the individual values in each group ( e , f , h ). d – f) PSAP and PGRN protein levels in the SFO were remarkably increased. Their quantification by densitometric analysis is represented in e for male and f for female, respectively. e ) For PSAP/GAPDH, two-way ANOVA revealed a significant main effects of genotype (F(1,24) = 547.7, p < 0.0001, ηp 2 = 0.48, 95 % CI [−74.51, −62.43]), with no effect of age ( p = 0.46) or genotype × age interaction ( p = 0.47). Post-hoc Tukey's tests showed that SAP-D −/− differed from WT at 3 M ( p < 0.0001, Cohen's d = 9.99, 95 % CI [−86.30, −54.96]), 6 M ( p < 0.0001, Cohen's d = 7.24, 95 % CI [−79.04, −47.70]), and 10 M ( p < 0.0001, Cohen's d = 8.77, 95 % CI [−87.09, −55.75]). For PGRN/GAPDH, two-way ANOVA revealed a significant main effect of genotype (F(1,24) = 354.2, p < 0.0001, ηp 2 = 0.48, 95 % CI [−46.39, −37.22]), with no effect of age ( p = 0.73) or genotype × age interaction ( p = 0.76). Post-hoc Tukey's tests showed that SAP-D −/− differed from WT at 3 M ( p < 0.0001, Cohen's d = 7.87, 95 % CI [−52.71, −28.91]), 6 M ( p < 0.0001, Cohen's d = 5.35, 95 % CI [−52.82, −29.02]), and 10 M ( p < 0.0001, Cohen's d = 8.77, 95 % CI [−52.32, −28.52]). f ) For PSAP/GAPDH, two-way ANOVA revealed a significant main effect of genotype (F(1,24) = 611.1, p < 0.0001, ηp 2 = 0.48, 95 % CI [−74.42, −62.95]), with no effect of age ( p = 0.07) or genotype × age interaction (p = 0.07). Post-hoc Tukey's tests showed that SAP-D −/− differed from WT at 3 M ( p < 0.0001, Cohen's d = 9.99, 95 % CI [−86.30, −54.96]), 6 M ( p < 0.0001, Cohen's d = 7.24, 95 % CI [−79.04, −47.70]), and 10 M ( p < 0.0001, Cohen's d = 8.77, 95 % CI [−87.09, −55.75]). For PGRN/GAPDH, two-way ANOVA revealed a significant main effect of genotype (F(1,24) = 273.5, p < 0.0001, ηp 2 = 0.47, 95 % CI [−63.96, −49.76]), with no effect of age ( p = 0.71) or genotype × age interaction (p = 0.70). Post-hoc Tukey's tests showed that SAP-D −/− differed from WT at 3 M ( p < 0.0001, Cohen's d = 7.31, 95 % CI [−73.43, −36.60]), 6 M ( p < 0.0001, Cohen's d = 5.71, 95 % CI [−73.04, −36.22]), and 10 M ( p < 0.0001, Cohen's d = 5.35, 95 % CI [−73.08, −36.26]). g – h ) Comparison of PSAP and PGRN protein expression in the SFO, whole cerebrum, and cerebellum. The quantitative analysis is shown in h . h ) For PSAP/GAPDH, one-way ANOVA revealed a significant effect in SAP-D −/− mice (F(2,6) = 30.06, p = 0.0007, η 2 = 0.90), but not in WT mice ( p = 0.3461). Tukey's post hoc tests showed significant differences for SFO versus cerebellum ( p = 0.0010, Cohen's d = 5.26, 95 % CI [46.80, 119.1]) as well as and cerebrum versus cerebellum ( p = 0.0018, Cohen's d = 9.34, 95 % CI [38.39, 110.6]). There was no significant difference for SFO versus cerebrum ( p = 0.76, Cohen's d = 0.47, 95 % CI [−27.71, 44.54]). For PGRN/GAPDH, one-way ANOVA revealed a significant effect in SAP-D −/− mice (F(2,6) = 54.42, p = 0.0001, η 2 = 0.94), but not in WT mice ( p = 0.6327). Tukey's post hoc tests showed significant differences for SFO versus cerebrum ( p = 0.0005, Cohen's d = 5.43, 95 % CI [26.19, 57.84]) as well as and SFO versus cerebellum ( p = 0.0005, Cohen's d = 5.43, 95 % CI [26.19, 57.84]), but not for cerebrum versus cerebellum ( p = 0.3274, Cohen's d = 6.06, 95 % CI [−7.72, 23.92]). ns: no significant difference. ∗∗∗∗ p < 0.0001. ∗∗∗ p < 0.001. ∗∗ p < 0.01.

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: Staining, Protein Extraction, Western Blot, Expressing, Comparison

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: Increased PSAP and PGRN immunostaining in the SFO and its surrounding tissues in SAP-D −/− mice a) Double immunofluorescent staining of PSAP (red) and PGRN (green) around the SFO in 10-month-old female WT and SAP-D −/− mice. The white dotted lines enclose the SFO. b ) enlarged white ⅰ-iv squares in a, as indicated. White arrowheads indicate co-staining with anti-PGRN and PSAP antibodies. Open arrowheads indicate staining with PGRN alone. Nuclei are labeled by DAPI (blue) staining. All scale bars, 20 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: Immunostaining, 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: 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: Lysosomal localization of PSAP and PGRN expression around the SFO a) Immunofluorescence staining of LAMP1 (green) around the SFO in 10-month-old female WT and SAP-D −/− mice. Nuclei are labeled by DAPI (blue) staining. b) Quantification of LAMP1-stained areas in ( a ) relative to WT (%). The results of Student's t-tests for each panel are as follows: left panel, p = 0.0059, Cohen's d = 2.35 (95 % CI: 15.89, 67.66); and right panel: p = 0.0061, Cohen's d = 2.33 (95 % CI: 51.06, 220.89). Data are shown as the mean ± SD (n = 5). c) Triple immunofluorescent staining of PSAP (red), PGRN (green), and LAMP1 (cyan) around the SFO of 10-month-old female SAP-D −/− mice. Enlarged images indicated by the white squares (ⅰ-iv) were shown in c. d) Localization rate of PGRN or PSAP to LAMP1 in the SFO. The results of Student's t-tests for each panel are as follows: left panel, p = 0.0008, Cohen's d = 2.24 (95 % CI: -40.90, -13.73); and right panel, p = 0.2341, Cohen's d = 0.64 (95 % CI: 26.80, 7.17). Data are shown as the mean ± SD (n = 6 for WT-SFO and n = 8 for SAP-D −/− -SFO). e) Same experiment as in (c) on microglia/macrophage co-expressing PSAP and PGRN, or PGRN only, in the boundary and fornix regions of SAP-D −/− mice. Single image for each antibody was shown in white, while double or triple merged images were presented with red (PSAP or PGRN), green (LAMP1 or PGRN), or cyan (LAMP1) as indicated. and indicate the individual values in each group (b and d). ns: no significant difference. ∗∗∗ p < 0.001. ∗∗ p < 0.01. All scale bars, 10 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, Immunofluorescence, Staining, Labeling

Journal: Npj Dementia

Article Title: Progranulin deficiency does not exacerbate TDP-43 pathology in TDP-43 transgenic mouse models

doi: 10.1038/s44400-025-00020-4

Figure Lengend Snippet: A Immunostaining of TDP-43 and NeuN in brain sections from 16-month-old mice WT, TDP-43 Q331K/Q331K (Q331K), Grn −/− , TDP-43 Q331K/Q331K Grn −/− (Q331K Grn −/− ) mice. Representative images from the cortex were shown. Scale bar, 10 µm. B Analysis of TDP-43, phosphorylated TDP-43 (pS409/410), and PGRN levels in cortical lysates from 16-month-old mice of the indicated genotypes. C TDP-43 levels in RIPA- and urea-soluble fractions were quantified. Data are presented as mean ± SEM from 3 mice per group ( n = 3). One-way ANOVA tests with Bonferroni’s multiple comparisons. D PGRN levels in RIPA soluble fractions were quantified and normalized to GAPDH. Data are presented as mean ± SEM from 3 mice per group ( n = 3). E Total RNAs were extracted from the cortex of 10-month-old WT, TDP-43 Q331K/Q331K , Grn −/− , TDP-43 Q331K/Q331K Grn −/− male mice, and the RT-qPCR was performed to analyze the splicing changes in Sort1 exon 17b (left) and Mapt exons 2 and 3 (right). The relative mRNA levels of transcripts including or excluding exons 2 and 3 represent the inclusion of Mapt exons 2 and 3. Data are presented as mean ± SEM ( n = 4 mice per genotype). p -values were determined using one-way ANOVA tests with Bonferroni’s multiple comparisons. F Expression levels of Tardbp in WT and Q331K mice. Total RNAs were extracted from the cortex of 10-month-old WT and Q331K male mice, and the RNA-seq was performed to analyze gene expression changes. Normalized read counts are shown. Data are presented as mean ± SEM ( n = 5-6 mice per genotype). * p < 0.05, unpaired two-tailed Student's t-test.

Article Snippet: The following antibodies were used in this study: rabbit anti-IBA-1 (Wako, 01919741), goat anti-AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-CD68 (Bio-Rad, MCA1957), mouse anti-GFAP (Cell signaling, 3670S), rabbit anti-TDP43 (Proteintech Group, 12892-1-AP (C-terminal) and 10782-2-AP (N-terminal)), rabbit anti-phospho-TDP-43 (Ser409/410) (Proteintech group, 80007-1-RR), mouse anti-PLP (Millipore, MAB388), mouse anti-MBP (Millipore, SMI-99), rabbit anti-MAG (Proteintech Group, 14386-1-AP), sheep anti-mouse PGRN (R&D systems, AF2557), and mouse anti-GAPDH (Proteintech Group, 60004-1-Ig).

Techniques: Immunostaining, Quantitative RT-PCR, Expressing, RNA Sequencing, Gene Expression, Two Tailed Test

Journal: Npj Dementia

Article Title: Progranulin deficiency does not exacerbate TDP-43 pathology in TDP-43 transgenic mouse models

doi: 10.1038/s44400-025-00020-4

Figure Lengend Snippet: A Immunostaining of TDP-43 and NeuN using rabbit anti-TDP-43 CTD antibodies and mouse anti-NeuN antibodies, respectively (left panel) or immunostaining of human TDP-43 (right panel) using mouse anti-human TDP-43 in brain sections from 21-day-old WT, hTDP-43 Tg/Tg (Tg/Tg), Grn −/− , and hTDP-43 Tg/Tg Grn −/− mice. Representative images from the cortex were shown. Scale bar, 10 µm. B Analysis of TDP-43 and phosphorylated TDP-43 (pS409/410) and PGRN levels in brain lysates from 21-day-old mice. Antibodies recognizing the C-terminal or N-terminal domain of TDP-43 were used to detect total TDP-43 levels. C TDP-43 and pTDP-43 levels in RIPA- and urea-soluble fractions were quantified. Data are presented as mean ± SEM from 3 mice per group ( n = 3). One-way ANOVA tests with Bonferroni’s multiple comparisons. ** p < 0.01. D PGRN levels in RIPA soluble fractions were quantified and normalized to GAPDH. Data are presented as mean ± SEM from 3 mice per group ( n = 3).

Article Snippet: The following antibodies were used in this study: rabbit anti-IBA-1 (Wako, 01919741), goat anti-AIF-1/Iba1 (Novus Biologicals, NB100-1028), rat anti-CD68 (Bio-Rad, MCA1957), mouse anti-GFAP (Cell signaling, 3670S), rabbit anti-TDP43 (Proteintech Group, 12892-1-AP (C-terminal) and 10782-2-AP (N-terminal)), rabbit anti-phospho-TDP-43 (Ser409/410) (Proteintech group, 80007-1-RR), mouse anti-PLP (Millipore, MAB388), mouse anti-MBP (Millipore, SMI-99), rabbit anti-MAG (Proteintech Group, 14386-1-AP), sheep anti-mouse PGRN (R&D systems, AF2557), and mouse anti-GAPDH (Proteintech Group, 60004-1-Ig).

Techniques: Immunostaining

Journal: Experimental Animals

Article Title: Progranulin deficiency attenuates tubulointerstitial injury in a mouse unilateral ureteral obstruction model

doi: 10.1538/expanim.23-0080

Figure Lengend Snippet: Progranulin (PGRN) and granulin (GRN) expression levels in unilateral ureteral obstruction (UUO)-operated kidneys. (A) mRNA expression levels of Grn in the kidneys of UUO-wild-type (WT) and Control-WT mice at days 3 and 7 after UUO. PGRN mRNA levels in the kidneys were higher in UUO-WT mice than in Control-WT mice. Comparisons among groups were performed using an unpaired t -test. *** P< 0.001. (B) Immunofluorescence staining for PGRN in kidney sections. PGRN expression was localized to the tubules. Blue and green indicate 4′,6-diamidino-2-phenylindole and PGRN, respectively. (C) PGRN and GRN protein levels in UUO-operated kidneys by western blotting. PGRN protein levels were significantly higher in the kidneys of UUO-WT mice on day 3 and 7 after UUO compared to those in Control-WT mice. Comparisons among groups were performed using an unpaired t -test. * P< 0.05. *** P< 0.001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KO, knockout.

Article Snippet: Kidneys were embedded in optimal cutting temperature compound, frozen, cut into 4- μ m sections using a cryostat, fixed in 4% fresh paraformaldehyde for 3 min, and blocked with 10% normal goat serum (Dako, Glostrup, Denmark) for 30 min. Immunohistochemical analyses were then performed using polyclonal sheep anti-PGRN antibodies (1:20 dilution in 1% bovine serum, AF2557; R&D Systems, Minneapolis, MN, USA) overnight at 4°C.

Techniques: Expressing, Control, Immunofluorescence, Staining, Western Blot, Knock-Out