Anti ldha

The IHC staining was used to examine the expression levels of c-Myc and glycolytic enzymes. The paraffin-embedded sections were incubated in a pressure cooker containing antigen retrieval buffer (pH 6.0) (Solarbio; G1202). Next, the slides were uniformly covered with 3% bovine serum albumin (Solarbio; A8020), and probed with the following primary antibodies prepared in a wet box at 4 °C overnight: anti-c-Myc (Servicebio; GB13076), anti-GLUT1 (Proteintech, 21,829–1-AP), anti-ENO1 (Proteintech, 11,204–1-AP), anti-PGK1 (Proteintech, 17,811–1-AP), anti-ALDOA (Proteintech, 11,217–1-AP), anti-HK1 (Proteintech, 19,662–1-AP), and anti-LDHA (Proteintech, 19,987–1-AP) antibodies. Next, the horseradish peroxidase (HRP)-labeled secondary antibody was used for 50 min. Then, the sections were then counterstained with hematoxylin.
The stained sections were scanned using Pannoramic MIDI and analyzed using Quant Center, which automatically identified all the strongly positive, moderately positive, weakly positive, and negative areas in the tissue sections. The H-scores were calculated as ∑ (percentage of intensity × intensity). The staining intensity was divided into three categories—strong, moderate, and weak—and the corresponding score was 3, 2, and 1, respectively.

1×10⁵ HEI-OC1 cells were seeded on cell climbing slices coated with poly-D-lysine in 24 well plates. When confluence was close to 60–80%, cell climbing slices were fixed with 4% paraformaldehyde for 20 min, permeabilized with 1% Triton X-100 for 10 min and then blocked in 10% goat serum for 1 h. Cells were then incubated with primary antibodies overnight. The primary antibodies included anti-GLUT1 (1:500, Abcam), anti-LDHA (1:250, Proteintech), and anti-HIF-1α (1:200, Abcam). The following morning, the cells were washed and incubated with CoraLite488-conjugated goat anti-rabbit/mouse IgG(H + L) (1:250, Proteintech) for 1 h. Cell climbing slices were then sealed with a mounting medium containing DAPI (Abcam, United States), and photographic images were acquired by confocal microscopy (Leica, Italy). Each experiment was repeated three independent times.

House dust mites (HDM, ALK-Abello A/S, Denmar), Recombinant Human long-isoform TSLP (lTSLP) was obtained from R&D systems. Synthetic sfTSLP peptides (63aa: MFAMKTKAALAI WCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ) were prepared by China Peptides (Shanghai, China). 3-PO (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one) and BAY 87-2243 (Selleck, China). Rabbit anti-HIF-1α, anti-LDHA, anti-PHD and anti-VHL (proteintech, China), Rabbit anti-STAT5, anti-p-STAT5, anti-JAK1, anti-p-JAK1, anti-JAK2and anti-p-JAK2 (Cell Signaling Technology, USA), Rabbit anti-TSLPR and mouse anti-IL-7R (santa curz, USA). Rabbit anti-TSLP (Abcam, USA).