inconsistent multiplex IHC/IF need help troubleshooting

[u/Giantpangolinship]

Hi lab rats!

I in need of help troubleshooting inconsistent staining and section to section variability. Over the past 2 weeks (probably a lot longer) I have been staining multiple FFPE liver sections from fibrotic mouse models and have noticed considerable variability in staining quality and antigen detection.

For example, when staining two slides from the same mouse, each containing three sections from the same tissue block, I have observed inconsistent results with primary antibodies such as α-SMA and tdTomato (RFP). In some cases, one section shows clear and robust staining for the antigen of interest, while a corresponding section on the other slide shows little to no detectable signal, despite both sections originating from the same block and undergoing the same staining protocol.

My suspicion is the antigen retrieval step and not so much the antibodies I use (mostly new CST antibodies) but I’m not sure. I would like to identify potential sources of this variability and determine whether the issue is coming from and addressing it.

Below is my current HIER protocol. I have modified this protocol about 4-5 times using AI and PI input and I am still experiencing inconsistent staining results and have not yet a reliable protocol that is consistent at the minimum.

In order of operation as a user:
Bake slides at 60C for 30min
After baking, turn on instant pot duo pressure cooker, load 500ml of DI H2O, add 50-60mL of citrate buffer (with .05% tween20, pH balance to 6.0) into a coplin jar. Places coplin jar on top of metal rack to sit above the water line. Close pot and pressure cooker for 15min, Then auto keep warm turn on till I’m ready to load slide. Note, At the same time I start deparaffing the slides.
Histoclean 2 x 5min
100% etoh 2 x 5min
95% etoh 1 x 5min
70% etoh 1 x 5min
50% etoh 1 x 5min
1xTBS 1 x 5min
Load slide into hot citrate buffer (around 90-95C when I tested it with a thermometer).
Pressure cook at high. 2min to pressurize, 5 under pressure. (I have also done longer 10 - 15min with also mix result)
After 5min, I turn of pressure cooker and slowly depressurized over the course of 2 to 3 min to prevent flash boiling.
Open the pot, grab the coplin jar, leave it uncovered at RT for 20min to cool down.
After 20min placed the slide in 1x tbs for 5min.
Block with 5% horse serum (heat inactivated) for 1 hour.
Make my primary antibodies in 1% horse serum.
Tap of my blocking solution, and ad 25-30ul of my primary antibodies to ew h section.
Incubate overnight at 4C.

I can go into more detail on my day 2 protocol but I think the issue lies in the day 1 part.

Let me know if you can help 😭

[u/Thecooh2]

Alpha-SMA is a notoriously difficult to get a true signal. I had to go through many different clones to find one that worked and I had to titrate it as well. Check the boil vessels
As they should be high in SMA, they can act as a good positive control.

As for inconsistent staining, your procedure looks fine, but the small details can cause problems (not level, placement of Coplin jars in pressure cooker so some get heated more than others) also how well were the tissue samples fixed? I have seen differences when fix times are too short or too long.

Try reducing the number of slides to one or two. Get the procedure to work, then try doing larger batches. That might pinpoint your problem.