Guide for collecting and reporting metadata on protocol variables and parameters from slide-based histotechnology assays to enhance reproducibility

Figures & data

Table 1. Guide for the collection of preanalytical slide-based metadata for transparent reporting of materials and methods.

Element	Element Type	Required Element Parameters	References
Sample source, tissue/cell type	Human, animal, xenograft, patient derived xenograft	Human tissue source, tissue block cohort, age, gender. Animal species and strain; embryo, developmental stage, cell line ^aRRID, genetic manipulation, gene construct, CRISPER, unique or novel sample type [gel, implant]	[94–102]
Sample preservation, preparation	Smear, touch-prep, FNA, freezing, electron microcopy	^bSample preparation: Fresh or fixed sample, sucrose cryoprotected; freezing method (LN2/isopentane, dry Ice/isopentane); freezer, cryostat ^b.c Sample preparation parameters. Fixation; TTF, TOF, fixative. Paraffin tissue processing (automated, manual, rapid, microwave); plastic; Number of cycles/steps, temperature °C, reagent concentration, pH	[98,99,103–111]
Embedding	Frozen, paraffin, plastic, cell block	^b,cCryoembedding media, paraffin, (melting point °C); ^dresin, plastic	[112–118]
Microtomy	Cryomicrotomy, FFPE microtomy, sledge, ultramicrotomy, vibratome, mineralized bone microtomy	^b,cType of microtomy (rotary, automated, sledge, cryostat, ultramicrotome), knife, blade, diamond, tungsten carbide). Section thickness; Cryotomy temperature (°C); water bath, slide section adhesive, section drying (°C) and time ^cSpecial methods: Tape transfer; saws, cutting/grinding system, radiography	[116,119–123]
Glass slide	Slide type and coating	^b,cCommercial coated slides; In-house coating	[124,125]
Mounted section storage	Storage conditions	Freezer, Temperature °C, humidity, time frame	[124–137]

^aRRID Resource Research Identifier (https://scicrunch.org/resources).

^bCommercial and Non-commercial reagents, chemicals, catalog/product numbers, vendor.

^cEquipment manufacturer, model, operating software version.

^d Epoxy resin, glycol methacrylate, methyl methacrylate; FNA fine needle aspirate, LN2 liquid nitrogen; TOF Time to fixation; TOF Time of fixation (duration); FFPE Formalin fixed paraffin embedded.

Table 2. Guide for collection of analytical slide-based metadata for transparent reporting of materials and methods.

Element	Element Type	Required Element Parameters	References
Manual, semi-automated, automated assay	Instrument/component manufacturer	Manufacturer, model, operating software version	[125,127,128,130,131,136,138–148]
Fixation (slide)	Frozen/fresh material	^a,bAir dry, freezer storage; fixatives, chemical (organic, cross-linking agents)	[115]
Deparaffinization	Manual, automated	^b,cManual, ^a,cautomated	[149–155]
Histochemistry	Routine diagnostic, special histochemical stains	^eMethod Name. ^aCommercial kits, ^bIn-house prepared stain reagents	[15,17,156–158]
Enzyme Histochemistry	Enzyme Histochemistry	^eMethod Name ^aCommercial kits, In-house prepared reagents, chemicals, pH; ^cECN	[19,159]
Buffers	Washing, rinsing	^aCommercial buffer; ^b,dIn-house prepared buffer, chemicals, pH
Antigen Retrieval	Automated, semi-automated, manual platforms; buffer	^aManual, semi-automated or automated IHC platform. (water bath, steamers, autoclave, pressure cooker, ^fmicrowave), ^a,b,dCommercial in-house prepared reagent.	[160–163]
Pretreatments	Enzymes	^cECN. ^aCommercial reagent kits. ^a, b, dIn-house reagents inclusive of combined or sequential applications	[149,164]
	Detergents	Detergent class (anionic, cationic, non-ionic, amphoteric) ^aCommercial reagent ^b,dIn-house preparation inclusive of combined or sequential applications	[164,165]
	Autofluorescence	^a,b,d,I Commercial kit, reagents. ^eMethods, In-house prepared reagents
Blocking Buffers	Non-specific, Endogenous component, Auto-fluorescence	^aCommercial reagent or kits, ^b,dIn house reagents inclusive of combined or sequential applications	[166]
Antibody Diluent	Buffers	^aCommercial reagent, ^b,d In-house prepared reagents, dilution, pre-incubation time	[166,167]
^kDetection Target Modality	^a,bAntibody (research use only (RUO) or in vitro diagnostic (IVD))	Species, clone, immunoglobulin, isotype, concentration and dilution, conjugation/tag, epitope, ^gUniProt number, ^hRRID	[168–170]
	^a,bProbe	^iEntrez Gene Identifier, ^a,b,d type (double-stranded or single stranded DNA, riboprobe, peptide-nucleic acid), probe production, vector, enzyme, label (isotopic, fluorescent, biotinylated, hapten), concentration, probe length, probe sequence (sense and anti-sense), denaturation hybridization buffer	[49,52–54,168–171]
	^a,bNanobody	Uniprot Number, target sequence, species, motif (scFv, Fab, F(ab)2), concentration, conjugation-tag	[172,173]
	^a,bAptamer	Aptamer type/motif (peptide, DNA, RNA, XNA) sequence, concentration, conjugation-tag	[174–178]
	^a,bLectins	Lectin name, source, ligand motif, conjugation-tag	[179]
Post-Treatments	Blocking, enzymes, detergents	^b,dCommercial reagent kits, In-house prepared reagents, ^cECN, Detergent class (anionic, cationic, non-ionic, amphoteric)	[165,166]
Detection Architecture	Traditional (direct, indirect, multiplex)	^a,b,d hCommercial detection kits and in-house prepared reagents with species, isotype, concentration, dilution, immunoglobulin specificity, species reactivity, conjugation/tag, enzyme reporter, multiplexing denaturation sequence and cycles	[180–187]
	Amplification (polymer, multimer, tyramide)	^a,b,d,hCommercial detection kits, In-house prepared reagents clonality, isotype, species, ligand-conjugation type, hapten, reporter enzyme	[188–191]
	In situ hybridization	^a.b.dCommercial detection kits and in-house prepared reagents Labeled complimentary DNA, RNA, branched DNA, hybridization chain reaction, rolling circle, isotopic, fluorescent, chromogenic, conjugation, signal amplification	[54]
Detection Chemistry	Chromogen, fluorophore	^a, b, d, hCommercial detection kits and In-house prepared reagents, chromogen, fluorescence excitation/emission wavelengths	[82,192,193]
Counterstain	Fluorescent, chromogenic Stain	^a,b,d,e,fMethod, steps, Commercial and non-commercial reagents and detection kits (a,b,d,i)	[165]
Post-Treatments	Autofluorescence	^a,b,d,hCommercial kit, reagents. ^eMethods, In-house prepared reagents,	[194,195]

^aCommercial reagents, kits, lot number, catalog number, equipment with make/model, manufacturer.

^bIn-house prepared reagents, chemicals, buffer composition, concentration, pH, catalog number, manufacturer.

^cECN Enzyme Commission Number.

^dIncubation time, temperature for each step or change, number of cycles or steps.

^eReference or method citation. ^fBiological Stain Commission dye certification number.

^fMicrowave wattage.

^gUniversal Protein Resource Knowledgebase Unique Identifier (https://beta.uniprot.org/).

^hRRID (https://scicrunch.org/resources).

ⁱNCBI Entrez Gene Identifier (https://www.ncbi.nlm.nih.gov/gene).

Table 3. Guide for the collection of post-analytical slide-based metadata for microscopy image capture.

Element	Element Type	Required Element Parameters	References
Microscope	Brightfield, fluorescence, phase contrast, confocal, multiphoton laser, transmission electron, DIC	^a ^bMicroscope, Light source; Objective type, magnification: Microscope filters, Fluorescence filter cubes	[58,60,196,197]
Camera	Analog SLR, Digital, Monochrome, Color, Multispectral	^a ^b,cCamera,Computer and operating system, monitor; Resolution, Image acquisition software and version, Image acquisition settings, Image acquisition functions, Image file format, Image compression format,	[58,60,61,66,74,196–201]
Whole Slide Scanner	Brightfield, Fluorescence, Multispectral	^a, b, cSlide Scanner; Computer and operating system, monitor, Image acquisition software and version, Magnification, Image acquisition settings, ICC profile, Image file format, Image compression format;	[66,72,74,78,196–202]

^aEquipment, make and model, manufacturer, country.

^bCalibration and quality control performed.

^cMicrons per pixel, DIC Differential Interference Microscopy. SLR Single Lens Reflex; ICC International Color Consortium.

Table 4. Microdissection and spatial transcriptomic techniques.

Technique	*Manufacturer	*Sampling Methodology	*Localization
Laser Capture Microdissection	Zeiss, Leica	Cryosection	Histochemical, IHC
GeoMx	Nanostring	Cryosection or FFPE	Oligo Barcoded Antibodies or Probe
10X Visium	10x Genomics	Cryosection or FFPE	mRNA Capture Microarray Slide
PhenoCycler (CODEX)	Akoya	Cryosection or FFPE	Oligo Barcoded Antibodies
Imaging Mass Cytometry	Fluidigm, IONPath	Cryosection or FFPE	Metal-labeled Antibodies
MALDI Imaging	Bruker	**Cryosection	Direct Metabolites

*See consideration for Elements, Element Type, Required Element Parameters in Table 1 and Table 2. **No cryoembedding media. FFPE Formalin Fixed Paraffin Embedded. IHC immunohistochemical.

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