Rabbit anti rat igg h l

Frozen tumor sections were stained with hematoxylin and eosin (Wako Pure Chemical Industries Ltd.). Collagen was stained using a Masson’s trichrome staining kit (25088-100, Polysciences, Inc.). For immunochemical staining, frozen tumor sections were fixed in 10% formalin for 10 min at room temperature. After washing with 1 × TBS, sections were incubated with 0.3% H₂O₂ in methanol at room temperature, followed by washing in 1 × TBS. To inhibit non-specific staining, sections were incubated in serum-free protein block solution (X0909, Dako) for 15 min at room temperature. The sections were then incubated with primary antibodies: rat anti-mouse CD31 monoclonal antibody (BD557355, BD Biosciences, 1:100) or rat anti-mouse F4/80 monoclonal antibody (MCA497, Bio-Rad, 1:100). After washing with 1 × TBS, signal stain boost IHC detection reagent (anti-rabbit, Cell Signaling Technology) or rabbit anti-rat IgG H&L (horseradish peroxidase conjugated) (ab6734, Abcam, 1:500) was added, and sections were incubated at room temperature for 30 min. After reaction with diaminobenzidine (8059S, Cell Signaling Technology), sections were counterstained with hematoxylin. A BZ-X710 microscope (Keyence, Osaka, Japan) was used for histologic observation and evaluation.

For immunohistochemical staining, sections were deparaffinized, rehydrated, and quenched for endogenous peroxidase activity with 0.3% H₂O₂, retrieved antigen with 0.01 M citrate buffer, and then blocked nonspecific binding sites with Tris-HCl containing 10% normal goat serum (NGS).
The sections were incubated overnight at 4 °C with primary antibody (CD3—Rabbit monoclonal antibody (1:250) (Thermo Fisher Scientific, Waltham, MA, USA; RM-9107-S0) (CD45—Purified Rat Anti-Mouse (1:50) (BD Bioscience, Franklin Lakes, NJ, USA; 550539) and (Iba1—Polyclonal rabbit antibody (1:400) (SynapticSystems, Göttingen, Germany, 234003) at an optimal concentration prepared in Tris-HCl containing 1% NGS. For CD 45, the slides were then incubated with Rabbit Anti-Rat IgG H&L (1:1000) (Abcam, Cambridge, UK; ab6703). The sections were incubated with HRP Labelled Polymer Anti-Rabbit Ig (Dako, Glostrup, Denmark; K 4010). Color development was performed using the 3,3′-diaminobenzidine substrate. All wash steps between incubations were with deionized water and Tris-HCl. The slides were counter-stained with hematoxylin, dehydrated, and mounted with mounting media. For the negative control, the same procedures were performed without adding the primary antibody.