Pano 7 plex ihc kit

Six pairs of lymph nodes were randomly selected, formalin-fixed, paraffin-embedded, and subjected to multiplex immunohistochemistry (mIHC). The mIHC was performed by using a PANO 7-plex IHC kit (Panovue, China) following the standard protocol [16 (link)]. Immune cell panels included the following antibodies: CD3 (1 : 200, Abcam, ab16669), CD8A (1 : 300, Cell Signaling Technology, 70306), Foxp3 (1 : 500, Abcam, 20034), PD1 (1 : 50, Cell Signaling Technology, 43248), and CD163 (1 : 100, Cell Signaling Technology, 93498). The slides were incubated with the primary antibodies, followed by 0.3% hydrogen peroxide solution for blocking endogenous peroxidase. DAPI (Sigma-Aldrich) was used for nuclear counterstaining. Images were acquired and analyzed by using a Mantra System (PerkinElmer) and inForm image analysis software (PerkinElmer), respectively.

Multiplex immunofluorescence staining of GD2, CD8, and CD163 in pre- and post-GD2 specific 4SCAR-T cell infusion specimens from Patient 01 was performed using a PANO 7-plex IHC kit (Panovue) as previously described [31 (link)]. After incubation with anti-GD2 (catalog no. 554,272; BD Biosciences), anti-CD8 (catalog no. 70,306; Cell signaling Technology), and anti-CD163 (catalog no. 93,498; Cell Signaling Technology) primary antibodies, horseradish peroxidase-conjugated secondary antibodies and a tyramide signal amplification fluorescence kit (Panovue) were applied. Nuclei were stained with DAPI, followed by scanning and multispectral images capture using the PanoVIEW vs200 slide scanner (Panovue), equipped with an Olympus 20×lens.

IHC was performed using an anti-CHAF1A antibody (ab126625), with a 2-step protocol. Specialized pathologists calculated the number of positively stained cells and the staining intensity to create grade categories under a microscope. A previously reported semi-quantitative method was used to assess IHC scores [22 (link)]. mIF staining was conducted using the PANO 7-plex IHC kit (Panovue, Beijing, China), section images were reconstructed using the Mantra System (PerkinElmer, Waltham, MA, USA), and quantification of cells in the images was performed using the inForm image software (PerkinElmer). Anti-CD8 (CST70306; Cell Signaling Technology, USA), anti-CD56 (CST3576), anti-CD68 (BX50031; Biolynx, China), anti-HLA-DR (ab92511), anti-panCK (CST4545), and anti-S100 (ab52642) antibodies were used for staining.

Lung samples obtained from infected and mock-infected mice were fixed with 4% paraformaldehyde, embedded in paraffin and cut into sections of 3.5 μm, and further used for TUNEL assay or processed to IHC. TUNEL assay was performed using an In Situ Cell Death Detection Kit (Roche) according to the manufacturer’s protocols. Briefly, the sections were deparaffinized, rehydrated, and permeabilized with proteinase K. The sections were then incubated with a mixture of TdT and dUTP (2 : 29), followed by incubation with DAPI (Beyotime, China) solution for 10 min. For IHC analysis, the indicated antibodies were used as primary antibodies, then sections were incubated with secondary antibody (Rabbit/Mouse Envision, Dako, Denmark), followed by visualizing with a detection kit (DAB, Dako, Denmark).
The biopsy lung tissue was fixed with 4% paraformaldehyde, paraffin-embedded and cut into sections of 5 μm. For routine histology, sections were stained with HE. Immunostaining was performed using PANO 7-plex IHC kit (Panovue, China) according to the manufacturer’s instructions. TUNEL assay was conducted as described above.

FFPE tissue sections were stained with the PANO 7‐plex IHC kit (Panovue) according to the manufacturer's instructions, including deparaffinisation with xylene, hydration with gradient ethanol, antigen retrieval with sodium citrate buffer (0.01 M, pH = 6.0), removal of endogenous peroxidase with H₂O₂, blocking tissues with 10% goat serum and staining with antibodies and TSA‐RM. The next cycle of staining started with antigen retrieval and was stopped with TSA labelling. The antibodies used included CD4 (ab133616, 1:500; Abcam) with Opal 620, CD8 (C8/144B, 1:200; CST) with Opal 690, and CD20 (ab78237, 1:2000; Abcam) with Opal 520. The antibodies used included CD45 (20103‐1‐AP, 1:2000; Proteintech) with Opal 570, SFRP4 (15328‐1‐AP, 1:200; Proteintech) with Opal 620, and IgG (EPR4421, 1:500; Abcam) with Opal 520. Then, all slides were scanned and analysed using the Vectra Automated Quantitative Pathology Imaging System (Vectra Polaris featuring MOTiF™). Finally, the percentage of SFRP4⁺ AFCs around the Tumour area was analysed in the HALO software (version 3.3). Specifically, the typical tumour area were identified based on DAPI staining, the distance and the area of 0–2, 2–4 and >4 mm from the tumour area were delineated, and the percentage of SFRP4⁺ AFCs in DAPI⁺ cells were calculated and statistical analysed.