Alexa fluor 488 conjugated rabbit anti rat igg

For the detection of PCNA58 (link), 4% PFA-fixed paraffin-embedded sections of back skin (5 μm thickness) were deparaffinized with toluene and rehydrated through a graded ethanol series. After inactivation of endogenous peroxidase with 0.3% H₂O₂ in methanol for 15 min and blocking with normal goat IgG, the sections were reacted overnight with the primary Ab against PCNA (PC10, Dako) in 1% BSA in PBS. After the reaction with HRP-conjugated goat anti-mouse IgG F(ab)’ second Ab, the sites of HRP were visualized with DAB and H₂O₂. For the detection of glomerular immune complex, kidney was embedded in OCT compound (Sakura Finetechnical) and frozen in liquid N₂. The tissue segments were sectioned with a cryostat at 5 μm. Frozen sections were fixed in cold acetone and blocked with 5% normal rat serum. The direct immunofluorescence technique was performed using Alexa Fluor 488-conjugated goat anti-mouse IgG(H+L) and Alexa Fluor 488-conjugated goat anti-Mouse IgM (μ chain) (Life Technologies). Alternatively, the frozen sections were stained with rat anti-mouse C3 (Abcam) followed by Alexa Fluor 488-conjugated rabbit anti-rat IgG (Life Technologies). Sections incubated with an appropriate isotype control primary Ab served as controls. The stained slides were analyzed as described above.

After the 4T1 cancer cell mouse model was treated with CXCL2 plasmid DNA (pDNA) in combination with HVJ-E treatment, HVJ-E treatment, CXCL2 pDNA treatment, or PBS treatment for 3 weeks, lung sections were fixed with 4% paraformaldehyde solution and blocked with 5% BSA. The sections were stained with Ultra-LEAF Purified anti-mouse Ly-6G antibodies (1A8, 127649, Biolegend). The secondary antibodies included an Alexa Fluor 488-conjugated rabbit anti-rat IgG (Life Technologies, Carlsbad, CA, USA). The sections were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and imaged with a confocal laser scanning microscope (LSM880 with Airyscan, Zeiss, Jena, Germany) equipped with the ZEN software program.