Among antibody selection for tissue imaging, the fixation process poses a challenge by epitope masking. Therefore, customized retriever step is required to expose these antigens for antibody binding. Additionally, tissue inherent autofluorescence can cause difficulties due to a high background staining.
However, the use of different approaches in the protocols in the literature hinders a standardized comparison between the images. Our study aims to address the demand for standardized procedures and assist the researcher in overcoming challenges during the imaging process. We established a versatile imaging protocol applicable to different tissues by comparing various different protocols.
So our work provides guiding and troubleshooting tips from antibody usage to sample mounting, highlighting the potential pitfalls. Our findings advance research by introducing a new primary antibody for citralinated Histon three and an autofluorescent reducing agent to minimize background staining. Furthermore, for successful imaging, we emphasize the careful handling of samples and the importance of maintaining a constant high temperature for antigen retrieval.
To begin, prepare 10 times concentrated citrate target retrieval solution or TRS of pH six, according to the manufacturer's instructions. Dilute it to a ratio of one to 10 with deionized water. Then heat the solution in a plastic staining jar to 96 degrees Celsius.
Remove the slides from the slide rack and place them into a wet chamber. Next pipette one or two drops of the autofluorescence reducing agent onto each tissue sample and incubate the samples for five minutes. Stack the slides back into the slide rack and rinse for one minute and 60%ethanol using upward and downward motions to remove the unused autofluorescence reducing reagent, submerge the slide rack in deionized water for five minutes.
Transfer the slides into a staining jar with preheated TRS buffer for antigen retrieval. Incubate the slides for 10 minutes at 96 degrees Celsius in a water bath. Remove the staining jar and let it cool to room temperature for approximately 60 to 90 minutes.
Next, remove the TRS while keeping the slides in the jar. Rinse the slides twice with tris buffered saline containing 0.05%tween at pH 7.4 for three minutes each to remove any remaining TRS residues, place the slides in a wet chamber, gently wipe excess water from the slides using paper tissues. Then encircle each sample using a hydrophobic barrier pen to prevent the antibody solution from running off.
Begin by pipetting one drop of ready-to-use blocking solution with donkey serum onto the tissue samples. Then incubate the samples for 30 minutes at room temperature, remove the excess blocking solution from the slides by tapping the edge against the hard surface. Dilute the primary antibodies in the antibody dilution buffer to prepare 100 microliters of the final solution for each sample.
Set up the antibodies, one tube for each primary antibody, and one for each ISO control antibody. Evenly spread 100 microliters of ISO control antibodies to one sample and 100 microliters of myelo peroxidase and citated histone H three or myelo peroxidase and neutrophil elastase antibody dilution to the other sample. Place the slides in a wet chamber and store them overnight at four degrees Celsius.
The following day, tap off excess primary antibody solution from the slides and stack them in a cuvete. Rinse the slides three times for five minutes each using trisespa saline with tween to remove the remaining primary antibody solution. Prepare two distinctly fluorescent secondary antibodies.
Donkey antigo for myelo peroxidase and donkey anti rabbit for citralinated histone H three staining. Dilute each antibody to 7.5 micrograms per milliliter concentration in the antibody dilution buffer. Place the slides in a wet chamber and dispense 100 microliters of the secondary antibody solution onto each sample.
Incubate them for 30 minutes at room temperature protected from light. Remove excess secondary antibody solution by tapping the slides. Put the slides in a slide rack, submerge the slides three times for five minutes each in a staining jar filled with PBS to wash off unbound antibodies.
Prepare the DAPI solution and dilute it to a concentration of one microgram per milliliter with deionized water. Submerge the slide drag in a staining jar containing the DAPI solution and incubate for five minutes at room temperature in the dark. Next, submerge the slide drag in a staining jar with PBS for five minutes to wash off any excess DAPI solution.
Finally, mount the samples using cover slips and mounting medium. The citralinated Histone H three antibody only bound to the extracellular histones and showed no staining of intracellular histones. The human or mouse specific myelo peroxidase antibody showed no specific staining.
While the universal human and mouse antibodies showed consistent good staining for myelo peroxidase. When no blocking agent was used, some mouse samples showed more nonspecific and partial positive staining. When blocked, the five minutes blocking time showed good results compared to the 10 minutes blocking time.
The higher temperatures in the microwave and water bath showed consistently moderate to good antigen retrieval. Whereas in a 60 degrees Celsius water bath there was only partially positive to no staining. For double staining, more favorable results were obtained for the samples incubated at 96 degrees Celsius Water bath, however, exceeding the incubation time of 40 minutes at 96 degrees Celsius resulted in less intense antibody staining.
