As I was seeing some structures that were not supposed to be there, first I tested if my antibodies has problems so I ran the experiment with WTB flies and primary antibodies with corresponding 2nd antibody

I used 3 predefined laser settings with minor changes. Alexa 488 uses laser at 488nm, Alexa 555 uses laser at 514 nm, Alexa 647 uses laser at 633nm

nc82 antibody (anti-mouse) and anti-mouse 650 ab. Image taken on green channel (488 excitation, 493-779 emission)

same sample but image taken on 555 wavelength:

same sample but image taken on 647 wavelength where I was supposed see the background staining. Its not clear because I did not adjust the laser power:

anti-RFP (rat) ab, anti-rat 555 staining:

image taken on 555 wavelength:

same sample but image taken on 647 wavelength:

Since the structure were visible with both of the antibodies, I suspected the reagents that I have been using might have contaminated so I prepared 4 different setups with no antibody staining at all:

Preparation 1: Includes prefixing the samples and blocking them with my existing NGS

Image taken with 488 laser and emission 493-737nm:

Same sample with 488 laser and emission 491-736nm

Same sample with laser 555 and emission 535-779:

Same sample with laser 647 and emission 638-779:

Preparation 2: Includes prefixing the samples and blocking them with aliquot from a new batch of NGS

Image taken with 488 laser and emission 493-744nm:

Image taken with 488 laser and emission 492-744nm:

Image taken with 555 laser and emission 535-756nm:

Image taken with 647 laser and emission 638-779nm:

Preparation 3: Includes prefixing the samples and no blocking with NGS:

Image taken with 488 laser and emission 493-739nm:

Image taken with 555 laser and emission 535-756nm:

Image taken with 647 laser and emission 638-779nm:

Preparation 4: No fixing and no blocking with NGS

Image taken with 488 laser and emission 493-744nm:

Image taken with 488 laser and emission 492-744nm:

Image taken with 555 laser and emission 535-779nm:

Image taken with 647 laser and emission 638-779nm: