Immunohistochemistry (Frozen sections) abreview for Anti-CCR4 antibody [KH-4F5]

Immunohistochemistry (Frozen sections)

Human Tissue sections (Frozen Section of Spleen, Tonsil and Normal Skin)

Yes - 0.3% Triton X-100

Frozen Section of Spleen, Tonsil and Normal Skin

Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C

Acetone

5 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: PBS

1/100

1/1000

Non-Abcam antibody was used: Goat anti-Mouse IgG1 Secondary Antibody, Alexa Flu
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor­ 594

I tested this antibody step-by-step by staining in Frozen tissue sections of Human Tonsil and Human Spleen as positive control tissue using various concentration dilutions [1/50 (6 ug/mL) 1/100 dilution (3 ug/mL) and 1/200 dilution (1.5 ug/mL)], varying the incubation time of the CCR4 antibody (5 hr vs overnight), using 0.3% Triton-X permeabilization buffer with 10% Normal Goat Serum during Blocking step. I also tried staining the antibody in frozen normal human skin tissue. (Attached is a image of CCR4 staining in frozen spleen tissue. Conditions: 1/100 dilution of primary Ab in PBS, overnight incubation in 4 degrees) Through various attempts at trying to optimize the antibody staining, I was unable to achieve any positive signal for CCR4+ cells in different frozen tissue sections (Human Tonsil, Human Spleen, Normal Human Skin).