Chicken anti gfap

Intestinal tissues were collected and fixed overnight in 4% paraformaldehyde. The fixed intestinal tissues were OCT-embedded, snap-frozen, and prepared into 16 μm sections for immunofluorescence staining. Cell samples including the primary EGCs, primary astrocytes and Caco-2 cells were seeded in 24-well plates plated with cell climbing slices respectively. After stimulation, cell samples were fixed in 4% paraformaldehyde and washed thrice with PBS for immunofluorescence staining. After permeabilization and blocking, the tissue sections or cell samples were incubated with the respective primary antibodies overnight at 4 °C. Briefly, the primary antibodies consisted of chicken anti-GFAP (1:200, GeneTex, Irvine, CA, USA), rabbit anti-Iba-1 (1:200 Abcam, Cambridge, UK), rabbit anti-Sox10 (1:500, Abcam, Cambridge, UK), anti-CD40 (1:200, Abcam, Cambridge, UK), and ZO-1 (1:200, Abcam, Cambridge, UK) antibodies. Next, the tissue sections or cell samples were washed thrice with PBS and incubated with A488 anti-chicken (1:1000, GeneTex) or A594 anti-rabbit (1:1000, GeneTex) antibodies at 37 °C for 2 h. DAPI (1:1000, Invitrogen) was used for nuclei counterstaining. An Olympus FluoView FV1000 microscope (Olympus, Tokyo, Japan) was used to collect the fluorescent images of intestinal tissues and cell samples. Fluorescent intensity was analyzed by ImageJ.

Intestinal tissues were collected and fixed overnight in 4% paraformaldehyde. The fixed intestinal tissues were OCT-embedded, snap-frozen, and prepared into 16 μm sections for immunofluorescence staining. Cell samples including the primary EGCs, primary astrocytes and Caco-2 cells were seeded in 24-well plates plated with cell climbing slices respectively. After stimulation, cell samples were fixed in 4% paraformaldehyde and washed thrice with PBS for immunofluorescence staining. After permeabilization and blocking, the tissue sections or cell samples were incubated with the respective primary antibodies overnight at 4 °C. Briefly, the primary antibodies consisted of chicken anti-GFAP (1:200, GeneTex, Irvine, CA, USA), rabbit anti-Iba-1 (1:200 Abcam, Cambridge, UK), rabbit anti-Sox10 (1:500, Abcam, Cambridge, UK), anti-CD40 (1:200, Abcam, Cambridge, UK), and ZO-1 (1:200, Abcam, Cambridge, UK) antibodies. Next, the tissue sections or cell samples were washed thrice with PBS and incubated with A488 anti-chicken (1:1000, GeneTex) or A594 anti-rabbit (1:1000, GeneTex) antibodies at 37 °C for 2 h. DAPI (1:1000, Invitrogen) was used for nuclei counterstaining. An Olympus FluoView FV1000 microscope (Olympus, Tokyo, Japan) was used to collect the fluorescent images of intestinal tissues and cell samples. Fluorescent intensity was analyzed by ImageJ.

Brain sections were washed 4× 10 min in Tris-buffered saline (TBS) at room temperature, followed by a 1-h blocking step in TBS²⁺ (TBS with 0.3% horse serum [Millipore] and 0.3% Triton X-100 [Sigma]) at room temperature. The tissue was transferred to 0.5 mL Safe Lock Reaction Tubes containing 200 μL TBS²⁺ including primary antibodies. Samples were incubated for 24−48 h at 4°C. Tissue samples were washed 4× 10 min in TBS at room temperature, followed by a 30-min blocking step in TBS²⁺ at room temperature. Brain sections were transferred to 0.5 mL Safe Lock Reaction Tubes containing 200 μl TBS²⁺ including secondary antibodies. Samples were incubated in the dark for 2 h at room temperature. Subsequently, brain slices were washed 4× 10 min in TBS at room temperature and were mounted on glass slides with Fluoromount G (eBioscience). The following antibodies were used: mouse anti-Sox2 (Abcam; 1:100), guinea pig anti-DCX (Merck; 1:400), rabbit anti-S100B (Abcam; 1:100), goat anti-mCherry (SICGEN; 1:1,000), and chicken anti-GFAP (GeneTex; 1:500). Nuclei were counterstained with Hoechst 33342 (BioTrend; 1:3,000).