TITLE: Immunofluorescence on FFPE tissue sections from inflamed and non-inflamed human gut 
AUTHORS: Ross J Porter, Katie J Smith, Gwo-Tzer Ho, Emily Gwyer Findlay

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Analysis of immunofluorescence is commonly confounded by autofluorescence and non-specific staining in the colorectum, particularly within highly inflamed samples. There is no consensus on how to definitively prevent this, and study protocols are variable. Our laboratory identified strong immunofluorescence within negative control samples stained for numerous target antigens, including CD3. Following methodical attempts to ensure there was no reagent contamination, we concluded that this was autofluorescence/ non-specific staining. Significant optimisation was subsequently performed. In this protocol, we report our optimised methodology for immunofluorescence experiments on formalin fixed paraffin embedded (FFPE) tissue sections, stained for identification of CD3+ lymphocytes. We hope this protocol acts as a template to help other laboratories optimise their own immunofluorescence protocols for similar antibodies.</div></div></div>

[STEPS]
?. [Deparaffinisation & Tissue Rehydration]
Sequentially immerse the slides, in the slide rack, into the following solutions:Xylene  Xylene 100% alcohol 100% alcohol 90% alcohol 80% alcohol 70% alcohol See image in Step 6 for the benefits of these enhanced timings.
?. [Deparaffinisation & Tissue Rehydration]
Place the slides immediately into tap water to prevent tissue dehydration. Never allow the tissue to become dehydrated.
?. [Heat-Induced Epitope Retrieval (HIER)]
Make sodium citrate retrieval buffer as below (adapted from Abcam):Dissolve   tri-sodium citrate (dihydrate) in    dH2OTitrate to  using 1N hydrochloric acidAdd  Tween®20 (Sigma-Aldrich, 9005-64-5)For our experiments, HIER buffer was made fresh each time (as 0.5-1L total was needed). However, Abcam suggest buffer is  stable for 3 months at room temperature, or longer at 4ºC.
2.94 g
1 L
0.5 µl
?. [Heat-Induced Epitope Retrieval (HIER)]
Heat the retrieval buffer, in a thermosetting plastic container, in the microwave (800W) at full power for .
[or until boiling/bubbling]
?. [Heat-Induced Epitope Retrieval (HIER)]
Immerse slides, in a slide rack, into the retrieval buffer. Microwave on full power for .At 7 minutes and 14 minutes add extra retrieval buffer to ensure slides remain completely immersed in liquid at all times. The benefits of enhanced deparaffinisation/tissue rehydration (Step 2) and enhanced HIER (Step 6), compared with a standard protocol (4 minutes in xylene / 10 minutes in the microwave), are illustrated below.
?. [Heat-Induced Epitope Retrieval (HIER)]
Cool slides in cold running tap water.
?. [Tissue Permeabilisation]
Immerse slides in 0.1% Triton X-100 (dissolved in PBS) for .
?. [Tissue Permeabilisation]
Wash with PBS Wash with PBS Wash with PBS
?. [Reduce Autofluorescence]
Make-up TrueBlack® Lipofuscin Autofluorescence Quencher 20X in DFM (Cat 23007, Biotium); make enough for . Vortex TrueBlack® Vortex to mix well
[per slide of 1:20 dilution as below]
?. [Reduce Autofluorescence]
Lay slides horizontally in a humidity/moisture chamber (see image at Step 15), wiping away any excess PBS with soft tissue. Do not dry the tissue.
?. [Reduce Autofluorescence]
Apply diluted  TrueBlack® solution to each slide for . Apply TrueBlack® to a maximum of 2-3 slides at a time, and use a stopwatch, to ensure accurate and consistent timing.DO NOT USE ANY DETERGENTS HEREAFTER; if absolutely essential then you should instead apply TrueBlack® before mounting - after step 31.
250 µl
?. [Blocking ]
Make the blocking solution. This should be PBS consisting of 25% donkey serum and 10% foetal calf serum.   Make enough for . We noted a significant decrease in background autofluorescence when both foetal calf serum and donkey serum were used in the blocking step compared with donkey serum alone.Donkey serum was used in this experiment because the secondary antibody was raised in donkey.
[per slide]
?. [Reduce Autofluorescence]
Wash with PBS Wash with PBS Wash with PBS Wash with PBS
?. [Blocking ]
Apply  of blocking solution to each slide and cover the whole slide gently with Parafilm®M, cut to shape, to ensure equal distribution of antibody and prevent tissue dehydration.
300 µl
?. [Primary Antibody Application]
Dilute the primary antibody/antibodies in 10% donkey serum diluted in 1x PBS - make enough for . DO NOT USE ANY DETERGENTS (e.g. Tween®), as this will remove TrueBlack® from the tissue.Example: Mouse monoclonal anti-human CD3 antibody (ab699 [GR3295418-1]) at 1:50
[per slide]
?. [Blocking ]
Lay slides horizontally in a humidity/moisture chamber, wiping away any excess PBS with soft tissue.
?. [Primary Antibody Application]
Tip excess block off each slide and lay horizontally in a humidity/moisture chamber.
?. [Blocking ]
Leave in a in a closed humidity/ moisture chamber, at  for .
0 Room temperature
?. [Primary Antibody Application]
Apply  of primary antibody solution to each slide and cover the whole slide gently with Parafilm®M, to ensure equal distribution of antibody and prevent tissue dehydration.
300 µl
?. [Primary Antibody Application]
Leave in a in a closed humidity/ moisture chamber, at .
[overnight]
?. [Secondary Antibody Application]
Wash with PBS Wash with PBS Wash with PBS
?. [Secondary Antibody Application]
Dilute the secondary antibody/antibodies in 10% donkey serum diluted in PBS - make enough for .Example: Donkey anti-mouse ab150108 at 1:500 (Alexa Fluor® 594)DO NOT USE ANY DETERGENTS (e.g. Tween®), as this will remove TrueBlack®.
[per slide]
?. [Secondary Antibody Application]
Lay slides horizontally in a humidity/moisture chamber, wiping away any excess block with soft tissue. Do not dry the tissue.
?. [Secondary Antibody Application]
Apply  of secondary antibody solution to each slide and cover gently with Parafilm®M, to ensure equal distribution of antibody and prevent tissue dehydration.
300 µl
?. [Secondary Antibody Application]
Leave in a in a closed humidity/ moisture chamber, for  at .Herein, protect the slides from light.
4 Room temperature
?. [DNA Staining]
Wash with PBS Wash with PBS Wash with PBS
?. [DNA Staining]
Make-up hoechst solution 1:1000, diluted in dH2O - make enough for . Protect this solution from light.
[per slide]
?. [DNA Staining]
Lay slides horizontally in a humidity/moisture chamber, wiping away any excess PBS with soft tissue. Do not dry the tissue.
?. [DNA Staining]
Apply  of hoechst solution to each slide for  at .
300 µl
0 Room temperature
?. [DNA Staining]
Wash with PBS Wash with PBS Wash with PBS
?. [Mounting]
Sequentially for each slide, wipe off excess PBS and mount a glass coverslip using  of anti-fade Floromount-Go® (SouthernBiotech 0100-01, L3220-ZD30) mounting medium.
150 µl
?. [Mounting]
Leave slides to dry on a slide tray, covered with aluminium foil (i.e. in complete darkness).
?. [Scanning & Analysis]
Digitally scan slides soon after staining (digital scanning profiles will need separate optimisation).
?. [Pre-Protocol Tissue Section Acquisition]
Slides should be clearly labeled with pencil and placed into a slide-rack.
?. [Scanning & Analysis]
Ensure the negative control is negative and positive control is positive (see image in Step 10).
?. [Scanning & Analysis]
Analyse objectively with a digital image-analysis software. QuPath (an open-source software) analysis works well for this application in our laboratory. Ensure you use the negative control to 'gate' out residual background staining/ autofluorescence.
?. [Further Optimisation Suggestions]
This protocol has been optimised using reagents in our laboratory to stain full thickness FFPE biopsies from inflamed and non-inflamed human gut specimens. We encourage further optimisation experiments in other laboratories, with different tissue sections and antibodies. Specific optimisation suggestions are listed below: Check there is no inadvertent damage occurring to tissue sectionsEnsure slides are not drying out, especially during antigen-retrieval in the microwave or during antibody incubationEnsure all timings and temperatures are accurate, especially during antigen retrievalBeware of chemical reagents that make tissue distortion or damage more likely (e.g. EDTA, Tween)Issues with antibody-epitope bindingAlter deparaffinisation and rehydration timesIncrease or decrease heat-induced epitope retrieval (HIER) timeTrial higher temperatures for HIER; consider use of a pressure cooker or autoclave Use a more accurate time and temperature controlled microwave (professional rather than household)Trial different reagents for HIER e.g. Tris-EDTA Trial different pH levels for HIER buffersReduce the time of primary antibody application (e.g. from overnight to 1 hour)Increase or decrease concentration of Tween (beware of increased tissue distortion at higher concentrations)Increase or decrease antibody concentrations (primary and secondary)If necessary, consider different fixation methods for your tissueTo optimise fluorescence and reduce autofluorescence/ non-specific background stainingEnsure you have robust positive and negative controls for every experimentIncrease the blocking time Increase the number and duration of washing stepsProtect slides from light during staining and imagingAttempt multiple staining steps (i.e. stain for each antibody sequentially)Alter the time slides are exposed to TrueBlack®  (e.g. decrease to 15s or increase to 1 minute)If there are issues with both antigen retrieval and fluorescence, try applying TrueBlack® at the end of this protocol before mounting (this will also allow increased use of detergents before this step) - see data sheet for TrueBlack®.  If TrueBlack®  is not effective for your tissues, consider other agents such as Vector®TrueVIEW® Autofluorescence Quenching Kit (SP-8400-15).Ensure the cover slip and mounting medium are optimal for your detection (and compatible with immunofluorescence)Ensure the best fluorophores are selected for imaging, and ensure there is good separation between fluorophores for co-stainsEnsure there are no dust or bubbles on the side at time of mountingIssues with antibody-epitope bindingAlter deparaffinisation and rehydration timesIncrease or decrease heat-induced epitope retrieval (HIER) timeTrial higher temperatures for HIER; consider use of a pressure cooker or autoclave Use a more accurate time and temperature controlled microwave (professional rather than household)Trial different reagents for HIER e.g. Tris-EDTA Trial different pH levels for HIER buffersReduce the time of primary antibody application (e.g. from overnight to 1 hour)Increase or decrease concentration of Tween (beware of increased tissue distortion at higher concentrations)Increase or decrease antibody concentrations (primary and secondary)If necessary, consider different fixation methods for your tissueTo optimise fluorescence and reduce autofluorescence/ non-specific background stainingEnsure you have robust positive and negative controls for every experimentIncrease the blocking time Increase the number and duration of washing stepsProtect slides from light during staining and imagingAttempt multiple staining steps (i.e. stain for each antibody sequentially)Alter the time slides are exposed to TrueBlack®  (e.g. decrease to 15s or increase to 1 minute)If there are issues with both antigen retrieval and fluorescence, try applying TrueBlack® at the end of this protocol before mounting (this will also allow increased use of detergents before this step) - see data sheet for TrueBlack®.  If TrueBlack®  is not effective for your tissues, consider other agents such as Vector®TrueVIEW® Autofluorescence Quenching Kit (SP-8400-15).Ensure the cover slip and mounting medium are optimal for your detection (and compatible with immunofluorescence)Ensure the best fluorophores are selected for imaging, and ensure there is good separation between fluorophores for co-stainsEnsure there are no dust or bubbles on the side at time of mountingTo optimise fluorescence and reduce autofluorescence/ non-specific background stainingEnsure you have robust positive and negative controls for every experimentIncrease the blocking time Increase the number and duration of washing stepsProtect slides from light during staining and imagingAttempt multiple staining steps (i.e. stain for each antibody sequentially)Alter the time slides are exposed to TrueBlack®  (e.g. decrease to 15s or increase to 1 minute)If there are issues with both antigen retrieval and fluorescence, try applying TrueBlack® at the end of this protocol before mounting (this will also allow increased use of detergents before this step) - see data sheet for TrueBlack®.  If TrueBlack®  is not effective for your tissues, consider other agents such as Vector®TrueVIEW® Autofluorescence Quenching Kit (SP-8400-15).Ensure the cover slip and mounting medium are optimal for your detection (and compatible with immunofluorescence)Ensure the best fluorophores are selected for imaging, and ensure there is good separation between fluorophores for co-stainsEnsure there are no dust or bubbles on the side at time of mountingIf there are issues with both antigen retrieval and fluorescence, try applying TrueBlack® at the end of this protocol before mounting (this will also allow increased use of detergents before this step) - see data sheet for TrueBlack®.  If TrueBlack®  is not effective for your tissues, consider other agents such as Vector®TrueVIEW® Autofluorescence Quenching Kit (SP-8400-15).Ensure the cover slip and mounting medium are optimal for your detection (and compatible with immunofluorescence)Ensure the best fluorophores are selected for imaging, and ensure there is good separation between fluorophores for co-stainsEnsure there are no dust or bubbles on the side at time of mountingIf the final staining results are inconsistent, consider use of an autostainer or optimising slide scanner settings.