Journal: Frontiers in Immunology

Article Title: Characteristics of tertiary lymphoid structures in prostate cancer and the impact of neoadjuvant therapy on their formation and maturation

doi: 10.3389/fimmu.2025.1663396

Figure Lengend Snippet: Clinicopathological features and prognostic significance of tertiary lymphoid structures (TLS) (A) The distribution of clinical features and immature density for the 27 patients in cohort 1. (B) Kaplan-Meier survival curves for progression-free survival (PFS) of 27 patients with no treatment before surgery stratified based on the median density of TLSs (B) , density of intra-/peri- TLSs (C) , maturation of TLSs (D) and the existence of SFL-TLSs (In SFL-TLS, CD21+ follicular dendritic cells form dense clusters within the core germinal center) (E) . B–E: Differentiate Kaplan-Meier curves with distinct colors for better visual clarity. 3D & 3E: Clarify the difference between “mature TLS” and “SFL-TLS.”.

Article Snippet: Then, slides were treated by microwave to induce antigen retrieval using citric acid solution for 15 min. 6 primary antibodies were used, including CD4 (1:500, #Ab67480, Abcam), CD8 (1:300, #66868-1-Ig, proteintech), CD20 (1:1000, #60271-1-Ig, proteintech), CD21 (1:200, #24374-1-AP, proteintech), Ki67(1:1000, #HA721115, HuaBio), pan-cytokeratin (1:2000, # HA601094 , HuaBio), DAPI (1:500, #C1002, Beyotime).

Techniques:

Journal: Frontiers in Immunology

Article Title: Characteristics of tertiary lymphoid structures in prostate cancer and the impact of neoadjuvant therapy on their formation and maturation

doi: 10.3389/fimmu.2025.1663396

Figure Lengend Snippet: Characteristics of tertiary lymphoid structures (TLS) in treatment-naive group and neoadjuvant hormone therapy (NHT) group in cohort 2. (A) mIHC of immature and mature TLS in NHT group and treatment-naive group in cohort 2; (B) Comparison of the ratio of CD4 + cells, CD8+ cells, CD20+cells and CD21+cells in tumor microenvironment (TME) in the NHT group and treatment-naive group. (C) Proportion of different TLSs maturation stage in the NHT group and treatment-naive group. (D) Proportion of different density of TLS in the NHT group and treatment-naive group. (E) Proportion of different location of TLS in the NHT group and treatment-naive group. P values are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001.

Article Snippet: Then, slides were treated by microwave to induce antigen retrieval using citric acid solution for 15 min. 6 primary antibodies were used, including CD4 (1:500, #Ab67480, Abcam), CD8 (1:300, #66868-1-Ig, proteintech), CD20 (1:1000, #60271-1-Ig, proteintech), CD21 (1:200, #24374-1-AP, proteintech), Ki67(1:1000, #HA721115, HuaBio), pan-cytokeratin (1:2000, # HA601094 , HuaBio), DAPI (1:500, #C1002, Beyotime).

Techniques: Comparison

Journal: Frontiers in Immunology

Article Title: Characteristics of tertiary lymphoid structures in prostate cancer and the impact of neoadjuvant therapy on their formation and maturation

doi: 10.3389/fimmu.2025.1663396

Figure Lengend Snippet: Characteristics and changes of immune cells in pre- and post- neoadjuvant hormone therapy (NHT) groups in GSE111177 . (A) Supervised clustering of PCa specimens by NHT (n = 24), displaying ssGSEA scores ( GSE111177 ). (B) The biological process of DEGs identified in pre-/post- NHT groups ( GSE111177 ). (C) Comparison of the expression of CD4, CD8A and CD8B, CD20, FOXP3, CD21, GZMB, PRF1 in PCa patients before and after NHT using GSE111177 cohort (n = 24). (D) Comparison of GSVA scores utilizing four TLS signatures in 24 paired pre-NHT and post-NHT samples from GSE111177 .

Article Snippet: Then, slides were treated by microwave to induce antigen retrieval using citric acid solution for 15 min. 6 primary antibodies were used, including CD4 (1:500, #Ab67480, Abcam), CD8 (1:300, #66868-1-Ig, proteintech), CD20 (1:1000, #60271-1-Ig, proteintech), CD21 (1:200, #24374-1-AP, proteintech), Ki67(1:1000, #HA721115, HuaBio), pan-cytokeratin (1:2000, # HA601094 , HuaBio), DAPI (1:500, #C1002, Beyotime).

Techniques: Comparison, Expressing

Journal: Frontiers in Immunology

Article Title: Characteristics of tertiary lymphoid structures in prostate cancer and the impact of neoadjuvant therapy on their formation and maturation

doi: 10.3389/fimmu.2025.1663396

Figure Lengend Snippet: Characterization of tumor immune microenvironment (TME) and tertiary lymphoid structures (TLS) before and after neoadjuvant hormone therapy (NHT). (A) Comparison of the ratio of CD4 + cells, CD8+ cells, CD20+cells and CD21+cells in TME before and after NHT. (B) Comparison of the TLS presence in TME before and after NHT. (C) Comparison of the intra-tumor TLS presence in TME before and after NHT. (D) Comparison of the TLS maturation stage in TME before and after NHT. (E) Evaluation of TLS maturation stage using mIHC staining of CD4, CD8 CD20, CD21 before and after NHT. P values are denoted as follows: **p < 0.01, ***p < 0.001, NS p>0.05.

Article Snippet: Then, slides were treated by microwave to induce antigen retrieval using citric acid solution for 15 min. 6 primary antibodies were used, including CD4 (1:500, #Ab67480, Abcam), CD8 (1:300, #66868-1-Ig, proteintech), CD20 (1:1000, #60271-1-Ig, proteintech), CD21 (1:200, #24374-1-AP, proteintech), Ki67(1:1000, #HA721115, HuaBio), pan-cytokeratin (1:2000, # HA601094 , HuaBio), DAPI (1:500, #C1002, Beyotime).

Techniques: Comparison, Staining

Journal: bioRxiv

Article Title: Tissue Kallikrein 1 cleaves complement factor C3 and activates the alternative complement pathway

doi: 10.1101/2025.09.08.674260

Figure Lengend Snippet: Citrated plasma (1 µL) from adult C57BL/6J mice injected with CsA + LPS (pre-injection [pre] and 24 hours post-injection [post]) was resolved on 4-15% SDS-PAGE under reducing conditions and immunoblotted for (A) KLK1 and KLK1+KAL, (B) FB and Ba, and (C) C3 and C3d. (A) Free and KAL-bound KLK1, (B) Ba and FB, and (C) C3d and C3 were quantified. Blue boxes indicate the quantified areas. MW = Molecular weight marker. F = female mouse, M = male mouse.

Article Snippet: These kidney sections then were stained with either biotinylated goat anti-mouse C3d antibody (20 μg/mL; R&D Systems, BAF2655) and streptavidin Alexa Fluor 488 conjugate (5 μg/mL; Invitrogen, S32354), following streptavidin/biotin blocking according to manufacturer’s instructions (Vector Laboratories, SP-2002) to block endogenous biotin and streptavidin binding sites, or goat anti-mouse TM antibody (10 μg/mL; R&D Systems, AF3894) and donkey anti-goat IgG Alexa Flour 488 (5 μg/mL; Invitrogen, A-11055).

Techniques: Clinical Proteomics, Injection, SDS Page, Molecular Weight, Marker

Journal: bioRxiv

Article Title: Tissue Kallikrein 1 cleaves complement factor C3 and activates the alternative complement pathway

doi: 10.1101/2025.09.08.674260

Figure Lengend Snippet: Adult C57BL/6J mice were injected intraperitoneally with CsA (30 mg/kg) + LPS (5 mg/kg). Citrated plasma was collected pre- and 24 hours post-CsA+LPS injection and analyzed by western blotting for KLK1, Ba, and C3d (C3b cleavage product); see Supplemental Figure 1. (A) Free and KAL-bound KLK1 were quantified and the bound fraction relative to the total (free + bound) was calculated. (B) Ba and FB were quantified and the percentage of Ba to total FB (FB + Ba) was calculated. (C) C3d and C3 were quantified and percentage of C3d to total C3 (C3 + C3d) was calculated. Columns = Mean, Error bars = SD, Blue = male, Pink = female. Significance was determined with a paired t-test. * = p <0.05, ** = p <0.01. (D) Ba and (E) C3d negatively correlated with KLK1 (Pearson correlation coefficient, r = −0.6684, p <0.05; Pearson correlation coefficient, r = −0.3914, p >0.2; respectively). (D and E) Gray = pre-CsA + LPS-injected mice, Black = 24 hours post-CsA + LPS-injected mice.

Article Snippet: These kidney sections then were stained with either biotinylated goat anti-mouse C3d antibody (20 μg/mL; R&D Systems, BAF2655) and streptavidin Alexa Fluor 488 conjugate (5 μg/mL; Invitrogen, S32354), following streptavidin/biotin blocking according to manufacturer’s instructions (Vector Laboratories, SP-2002) to block endogenous biotin and streptavidin binding sites, or goat anti-mouse TM antibody (10 μg/mL; R&D Systems, AF3894) and donkey anti-goat IgG Alexa Flour 488 (5 μg/mL; Invitrogen, A-11055).

Techniques: Injection, Clinical Proteomics, Western Blot

Journal: bioRxiv

Article Title: Tissue Kallikrein 1 cleaves complement factor C3 and activates the alternative complement pathway

doi: 10.1101/2025.09.08.674260

Figure Lengend Snippet: Adult C57BL/6J mice were injected intraperitoneally with CsA (30 mg/kg) + LPS (5 mg/kg) or DMSO (control). Citrated plasma and kidneys were collected 24 hours post-injection. Mounted fixed frozen kidney sections were incubated with biotinylated anti-mouse C3d (C3b cleavage product)/streptavidin Alexa Fluor 488 (green, A and C ) or anti-mouse TM/anti-goat IgG Alexa Flour 488 (green, B and D ). Cell nuclei were stained with DAPI (blue). Stained kidney sections were imaged by immunofluorescence microscopy with a 60x objective. Scale bar = 10 micrometers. Plasma sTM was measured by ELISA (E) , and circulating creatinine was measured by enzymatic assay (F) . Columns = Mean, Error bars = SD, Blue = male, Pink = female. Significance was determined with an unpaired t-test. * = p <0.05, ** = p <0.01. (G) sTM positively correlated with creatinine in DMSO- (●) and CsA + LPS-treated (▲) mice. Pearson correlation coefficient, r = 0.8326, p <0.05.

Article Snippet: These kidney sections then were stained with either biotinylated goat anti-mouse C3d antibody (20 μg/mL; R&D Systems, BAF2655) and streptavidin Alexa Fluor 488 conjugate (5 μg/mL; Invitrogen, S32354), following streptavidin/biotin blocking according to manufacturer’s instructions (Vector Laboratories, SP-2002) to block endogenous biotin and streptavidin binding sites, or goat anti-mouse TM antibody (10 μg/mL; R&D Systems, AF3894) and donkey anti-goat IgG Alexa Flour 488 (5 μg/mL; Invitrogen, A-11055).

Techniques: Injection, Control, Clinical Proteomics, Incubation, Staining, Immunofluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Enzymatic Assay

Journal: Kidney international

Article Title: Streptococcal pyrogenic exotoxin B antibodies in a mouse model of glomerulonephritis.

doi: 10.1038/sj.ki.5002407

Figure Lengend Snippet: Figure 4 | Complement activation in the kidney of mice hyperimmunized with 28-kDa SPE B mutant protein C192S. BALB/c mice were immunized four times with (a) BSA or (b) C192S. Complement C3 deposits were detected using FITC-conjugated anti-C3d antibody with fluorescence microscopy (n ¼ 10 per group). (c) The mean integral fluorescent intensity of glomeruli was analyzed in 10 fields per mouse (n ¼ 10 mice). Original magnification 200.

Article Snippet: Complement C3 was detected using sheep anti-human C3d (Serotec Ltd), which cross-reacts with mouse C3d, and then using FITC-conjugated goat anti-sheep IgG antibody (Jackson ImmunoResearch Laboratories Inc.).

Techniques: Activation Assay, Mutagenesis, Fluorescence, Microscopy

Journal: Kidney international

Article Title: Streptococcal pyrogenic exotoxin B antibodies in a mouse model of glomerulonephritis.

doi: 10.1038/sj.ki.5002407

Figure Lengend Snippet: Figure 8 | Immunoglobulin deposition and complement activation in mice after passive immunization with monoclonal anti-SPE B antibody 10G. BALB/c mice were intravenously immunized each day with 500 mg of monoclonal 9C or 10G IgG for 3 days and, after 7 days, the kidney sections were analyzed using (a, c) FITC-conjugated anti-mouse IgG or (b, d) anti-C3d antibody (n ¼ 3 per group). Original magnification 200.

Article Snippet: Complement C3 was detected using sheep anti-human C3d (Serotec Ltd), which cross-reacts with mouse C3d, and then using FITC-conjugated goat anti-sheep IgG antibody (Jackson ImmunoResearch Laboratories Inc.).

Techniques: Activation Assay

Journal: OncoTargets and Therapy

Article Title: Correlation of C-X-C chemokine receptor 2 upregulation with poor prognosis and recurrence in human glioma

doi: 10.2147/ott.s91626

Figure Lengend Snippet: Figure 1 High level of CXCR2 expression shown in cases of high-grade gliomas. Notes: (A) Representative sections for CXCR2 immunohistochemistry (IHC, SP ×400). (a) Normal nontumorous tissue, (b) WHO low-grade glioma, (c) WHO high-grade glioma, (d) negative control for immunostaining. Positive CXCR2 staining = brown; cell nuclei = blue; the arrows show representative results of staining. (B) The population of cells with different levels of CXCR2 expression in glioma and control brain tissue. Overall, the level of CXCR2 expression was significantly higher in WHO III–IV gliomas tissues than in WHO I–II glioma tissues and the control brain tissues according to IHC results. (C) Western blotting of CXCR2 protein level in gliomas and normal tissue. The upper panel is a representative result of Western blotting. CXCR2 protein expression was calculated by normalizing CXCR2 intensity to GAPDH intensity, and data were compared to the normal tissue, represented as 1. Data are expressed as mean ± SD; *P,0.05 versus normal tissue; #P,0.05 between different grades. Abbreviations: CXCR2, C-X-C chemokine receptor 2; WHO, World Health Organization; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation.

Article Snippet: Western blotting was performed according to standard protocols using the following antibodies: goat anti-glyceraldehyde 3-phosphate dehydrogenase polyclonal antibody (1:800; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-human CXCR2 monoclonal antibody (1:500); donkey anti-goat IRDye (1:10,000; Rockland, Limerick, PA, USA), and donkey anti-mouse IRDye (1:10,000; Rockland).

Techniques: Expressing, Immunohistochemistry, Negative Control, Immunostaining, Staining, Control, Western Blot, Standard Deviation

Journal: OncoTargets and Therapy

Article Title: Correlation of C-X-C chemokine receptor 2 upregulation with poor prognosis and recurrence in human glioma

doi: 10.2147/ott.s91626

Figure Lengend Snippet: Figure 2 CXCR2 inhibitor SB225002 could reduce cell migration. Notes: (A) Representative images for wound healing assay. U251 cells treated with DMSO or with SB225002 (400 nM) at the different time points of 0 hour, 12 hours, 24 hours, 36 hours, and 48 hours, respectively (bar 100 µm). (B) Statistical results of the wound healing assay. Images were analyzed using ImageJ analysis software and data presented as average length of cell-free void ± SD (*P,0.05). Abbreviations: CXCR2, C-X-C chemokine receptor 2; DMSO, dimethyl sulfoxide; SD, standard deviation; h, hour.

Article Snippet: Western blotting was performed according to standard protocols using the following antibodies: goat anti-glyceraldehyde 3-phosphate dehydrogenase polyclonal antibody (1:800; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-human CXCR2 monoclonal antibody (1:500); donkey anti-goat IRDye (1:10,000; Rockland, Limerick, PA, USA), and donkey anti-mouse IRDye (1:10,000; Rockland).

Techniques: Migration, Wound Healing Assay, Software, Standard Deviation