CXCL13 Antibody (Rabbit mAb) [E12F15] Datasheet

Biological Description

Specificity	CXCL13 Antibody (Rabbit mAb) [E12F15] detects endogenous levels of total CXCL13 protein.
Background	CXCL13, originally identified as B-cell-attracting chemokine 1 (BCA-1), belongs to the CXC subfamily of chemokines and acts through a single cognate receptor, CXCR5, forming a dedicated ligand-receptor axis that governs the architecture of secondary lymphoid organs (SLOs) such as lymph nodes, spleen, and Peyer's patches.CXCL13 and its receptor, CXCR5, make crucial contributions to this process by triggering intracellular signaling cascades in malignant cells and modulating the complex tumor microenvironment in an autocrine or paracrine fashion. Under physiological conditions, the same axis directs naive B cells and CXCR5-expressing T follicular helper (Tfh) cells toward B-cell follicles. CXCL13 is secreted by follicular helper T cells, follicular dendritic cells, and marginal reticular cells, and it recruits peripheral CXCR5-positive B cells into the B-cell zone or germinal center through high endothelial venules, where CXCL13 enhances follicle development and sustains secondary lymphoid organ homeostasis through a positive-feedback loop with B cells and follicular dendritic cells. Ligand engagement dissociates the receptor-coupled heterotrimeric G protein into Gα and Gβγ subunits, initiating distinct downstream cascades that translate the chemokine gradient into directional migration and cytoskeletal reorganization. CXCR5 enables B cells and Tfh cells to follow CXCL13 gradients toward B-cell follicles, establishing the spatial proximity required for productive T cell–B cell interaction, and naive T cells primed by dendritic cells generate CXCR5-positive pre-Tfh cells that migrate along CXCL13 gradients toward follicles, where interactions with cognate B cells license their entry and maturation into fully programmed B-cell helpers. This positioning underlies the generation of high-affinity antibody responses, since Tfh cells are essential contributors to B-cell proliferation, differentiation, and high-affinity antibody synthesis and are required for germinal center formation and maintenance. The axis extends adaptive immune organization into non-lymphoid tissue: CXCL13/CXCR5 activities organize and shape adaptive B-cell-related immune responses outside secondary lymphoid organs through the formation of ectopic lymphoid structures in inflamed tissues, including the central nervous system, and correspondingly, CXCL13 is proposed to recruit B cells to the cerebrospinal fluid during neuroinflammation. In malignancy, receptor engagement reshapes the tumor microenvironment through recruitment of both effector and suppressive populations, so that the CXCL13/CXCR5 axis has a dominant role in B-cell recruitment and tertiary lymphoid structure formation, activating immune responses against some tumors, while in most cancer types the same axis mediates pro-neoplastic immune reactions by recruiting suppressive immune cells into tumor tissue. Disruption of the axis at the developmental level produces measurable structural consequences, as CXCL13/CXCR5-deficient mice show incomplete maturation of lymph nodes. In contrast, overexpression of the pathway causes ectopic lymphoid structures to develop in non-lymphoid tissue.

Specificity

CXCL13 Antibody (Rabbit mAb) [E12F15] detects endogenous levels of total CXCL13 protein.

Background

CXCL13, originally identified as B-cell-attracting chemokine 1 (BCA-1), belongs to the CXC subfamily of chemokines and acts through a single cognate receptor, CXCR5, forming a dedicated ligand-receptor axis that governs the architecture of secondary lymphoid organs (SLOs) such as lymph nodes, spleen, and Peyer's patches.CXCL13 and its receptor, CXCR5, make crucial contributions to this process by triggering intracellular signaling cascades in malignant cells and modulating the complex tumor microenvironment in an autocrine or paracrine fashion. Under physiological conditions, the same axis directs naive B cells and CXCR5-expressing T follicular helper (Tfh) cells toward B-cell follicles. CXCL13 is secreted by follicular helper T cells, follicular dendritic cells, and marginal reticular cells, and it recruits peripheral CXCR5-positive B cells into the B-cell zone or germinal center through high endothelial venules, where CXCL13 enhances follicle development and sustains secondary lymphoid organ homeostasis through a positive-feedback loop with B cells and follicular dendritic cells. Ligand engagement dissociates the receptor-coupled heterotrimeric G protein into Gα and Gβγ subunits, initiating distinct downstream cascades that translate the chemokine gradient into directional migration and cytoskeletal reorganization. CXCR5 enables B cells and Tfh cells to follow CXCL13 gradients toward B-cell follicles, establishing the spatial proximity required for productive T cell–B cell interaction, and naive T cells primed by dendritic cells generate CXCR5-positive pre-Tfh cells that migrate along CXCL13 gradients toward follicles, where interactions with cognate B cells license their entry and maturation into fully programmed B-cell helpers. This positioning underlies the generation of high-affinity antibody responses, since Tfh cells are essential contributors to B-cell proliferation, differentiation, and high-affinity antibody synthesis and are required for germinal center formation and maintenance. The axis extends adaptive immune organization into non-lymphoid tissue: CXCL13/CXCR5 activities organize and shape adaptive B-cell-related immune responses outside secondary lymphoid organs through the formation of ectopic lymphoid structures in inflamed tissues, including the central nervous system, and correspondingly, CXCL13 is proposed to recruit B cells to the cerebrospinal fluid during neuroinflammation. In malignancy, receptor engagement reshapes the tumor microenvironment through recruitment of both effector and suppressive populations, so that the CXCL13/CXCR5 axis has a dominant role in B-cell recruitment and tertiary lymphoid structure formation, activating immune responses against some tumors, while in most cancer types the same axis mediates pro-neoplastic immune reactions by recruiting suppressive immune cells into tumor tissue. Disruption of the axis at the developmental level produces measurable structural consequences, as CXCL13/CXCR5-deficient mice show incomplete maturation of lymph nodes. In contrast, overexpression of the pathway causes ectopic lymphoid structures to develop in non-lymphoid tissue.

Usage Information

Application

IHC

Dilution

IHC

1:500

Reactivity

Mouse

Source

Rabbit Monoclonal Antibody

MW

13 kDa

Storage Buffer

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage
(from the date of receipt)

-20°C (avoid freeze-thaw cycles), 2 years

Experimental Methods
IHC	Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

Experimental Methods

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

References

https://pubmed.ncbi.nlm.nih.gov/34947813/
https://pubmed.ncbi.nlm.nih.gov/36078057/

Application Data

IHC

Validated by Selleck

Immunohistochemical analysis of formalin fixed paraffin embedded mouse spleen tissue with F9472 at 1:500 dilution.