I'm doing an IF experiment to show the GBP (guanylate binding protein) protein level (cytosolic protein).

However I'm having trouble doing my experiment, the fluorescence signals are expressed in the background, not at the protein location.

Other papers staining the GBP protein show that only specific areas with GBP are stained like dots.

The protocol below was used to stain the GBP protein in the raw cell line.

1. Fixation: Incubate the sample with 4% PFA for 15min at RT. Wash in PBS 3 times.

2. Permeabilization: Incubate the samples for 10min with 0.1% PBST(0.1% Triton X-100 in PBS). Wash in PBS 3 times.

3. Blocking: Incubate the cell with 5% goat serum with 0.1% PBST for 40min and wash 3 times in 1% goat serum (PBS).

4. Staining: Dilute 1’ Ab in 1% goat serum in PBS and stain at 4’ for O/N and wash in 1% goat serum with PBS 3 times. Dilute the 2’ Ab in 1% goat serum in PBS and incubate at RT for 1h in the dark. Wash in 1% goat serum with PBS.

5. Nuclear counterstaining and mounting: Incubate the antifade DAPI solution for 2-4min at RT.

I am using these antibodies,

1. GBP1-5 (Santa Cruz, sc-166960): dilution 1:200

2. Goat anti-mouse IgG (H+L) Highly Cross-adsorbed secondary antibody Alexa Fluor 488 (Invitrogen): dilution 1:400

Which steps do you think are creating the wrong staining image?

I attached the image that I made.

Thank you.