Goat anti mouse igg h l

The esophageal histological analysis using HE staining followed the established standard procedures^{51 (link)}. IF staining was performed to examine the homing and differentiation of infused DPSCs in the esophageal tissue^{52 (link)}. Sections of the esophagus were incubated in PBS containing 5% normal donkey serum and mouse anti-PCNA antibody (1:100, Santa Cruz Biotechnology, BM0104), mouse anti-CK14 antibody (1:100, Abcam, ab49747), mouse anti-integrin α6 antibody (1:100, Santa Cruz Biotechnology, sc-13542) and mouse anti-CD71 antibody (1:200, AbD, MCA155R) overnight at 4 °C. Sections were then washed with PBS and subsequently reacted for 1 h with goat anti-mouse IgG (H + L; 1:200, Earthox, E032220) in a box with a constant temperature of 37 °C. Sections were re-dyed with DAPI (Vector Laboratories, California, USA), washed, and mounted with an anti-fading medium before undergoing microscopic observations with a laser confocal microscope.

To identify the ΔNcGRA6 strain at the protein level, we extracted the protein from the ΔNcGRA6 strain and Nc-1 strain to detect NcGRA6 and NcSRS22A protein levels via western blot analysis. A rat polyclonal antibody against NcGRA6 and a mouse monoclonal antibody against the putative surface antigen NcSRS22A were prepared and constructed in our laboratory. First, the purified ΔNcGRA6 strain and Nc-1 strain tachyzoites were lysed using RIPA buffer (Beyotime, China) supplemented with 1 mM PMSF protease inhibitor. Then, the lysates were mixed with 5 × SDS-PAGE Sample Loading Buffer (Beyotime, China), boiled for 5 min, and electrophoresed on a 12% (w/v) SDS-PAGE gel. After electrophoresis, the protein bands were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) using a Mini Trans-Blot® 25Electrophoretic Transfer Cell (BIO-RAD, USA). The membranes were blocked with 5% (w/v) skim milk at 4°C overnight, incubated with primary antibody targeting NcGRA6 or NcSRS22A (1:200) for 2 h at 37°C, followed by incubation with HRP-conjugated secondary goat anti-rat IgG (H+L) or goat anti-mouse IgG (H+L) (Earthox, USA, 1:5,000) for 1 h at 37°C, respectively. Finally, the bands were detected using ECL chemiluminescence reagents (Thermo, USA) and visualized with an ECL western blot Detection System (Clinx Science Instruments Co., Ltd., China).

For double-immunofluorescence staining, the slides were routinely baked, dewaxed, and hydrated as previously described [27 (link), 28 (link)]. After antigen retrieval using hot citrate buffer (pH = 6), the slides were incubated with a blocking reagent (goat serum, Boster) for 1 h at room temperature. SCLC sections were incubated with the following primary antibodies: mouse anti-NSE monoclonal antibody (1:150; Proteintech) and rabbit anti-NBL1 polyclonal antibody (1:200; Affinity) overnight at 4 °C. The sections were then incubated with secondary antibodies for 2 h at room temperature after thorough washing, followed by staining with DAPI staining solution (Beyotime) for 15 min. The secondary antibodies used were Dylight488, goat anti-mouse IgG (H + L) (Earthox) and Dylight649, and goat anti-rabbit IgG (H + L) (Earthox). Sections were mounted using an Antifade reagent (Applygen) for fluorescence microscopy.

Cells were washed twice with ice-cold 1×PBS and then lysed in RIPA buffer (Applygen Technologies) containing protease inhibitor cocktail (Amresco). Cell lysates were then centrifuged for 15 min at 15,000×g at 4 °C, and the insoluble debris was discarded. Protein concentration was determined by using a BCA protein assay reagent (Pierce). Cell lysates (20–40 μg) were subjected to 8–15% SDS-PAGE and transferred to nitrocellulose membranes (Millipore). The membrane was blocked using 5% milk in TBST buffer at room temperature for 1 h. Primary antibodies were blotted using 5% milk or BSA in TBST, and incubated at 4 °C overnight. Secondary antibodies (Dylight 800, Goat Anti-Rabbit IgG (H+L) (EarthOx, E032820) or Goat Anti-Mouse IgG (H+L) (EarthOx, E032810)) were incubated for 1 h at room temperature in 5% milk/TBST. Then the signals were captured by the Odyssey system.