MID1 / E3 ubiquitin-protein ligase MID1 · IHC design guide

Design Immunohistochemistry for MID1

Plan MID1 IHC in paraffin sections around cytoplasmic staining in glandular, glial and renal tubular cells (HPA tissue IHC). Start the catalog antibody at 0.5–1 µg/mL (datasheet A01774-1) and include a no-primary control (standard IHC practice).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for MID1 (IHC for MID1): expected localisation Cytoplasmic tissue staining (HPA tissue IHC), antibody A01774-1, validated IHC image, and IHC protocol steps — Printable **MID1** IHC protocol sheet — expected localisation Cytoplasmic tissue staining (HPA tissue IHC), antibody A01774-1, controls and protocol steps. Open the full MID1 IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

MID1 Immunohistochemistry Experimental Design Guide

Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.

Must know before staining

	Expected localisation	Cytoplasmic tissue staining (HPA tissue IHC)
	Staining pattern	Cytoplasmic staining in glandular, glial and renal tubular cells (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet A01774-1)
	Positive control ⓘ	Appendix+4 more · see all
	Negative control ⓘ	Adipose tissue+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01774-1)
	Caveat	Antibody staining and RNA show low consistency (HPA tissue IHC)
	Regulation	Adult heart, placenta and brain are most abundant (UniProt)
	Isoform / epitope	2 isoforms; confirm epitope coverage (UniProt)

Section 1

Recommended MID1 IHC & IF Protocols

The catalog antibody uses citrate pH 6 retrieval (datasheet: A01774-1). The published IHC protocols below describe temporal cortex and prostate specimens (PMC5653760; PMC4074869: IHC methods).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human placenta tissues; fixative not specified (datasheet A01774-1)
Fixation	Image fixative and duration unreported (datasheet A01774-1); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Citrate pH 6, 20 min (datasheet A01774-1)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A01774-1)
Primary antibody	Rabbit anti-MID1, 0.5-1μg/ml (datasheet A01774-1)
Primary incubation	Overnight at 4 °C (datasheet A01774-1)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01774-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	MID1-positive staining in glandular cells of appendix (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in several tissues. No signal in the no-primary control.

💡Decision noteStart with citrate pH 6 for the catalog antibody (datasheet: A01774-1). The published methods used pepsin or CC1 retrieval with their own antibodies (PMC5653760; PMC4074869: IHC methods).

Section 2

What Is the Expected MID1 Staining Pattern?

MID1 is a cytoplasmic, microtubule-associated protein with no transmembrane segment (UniProt O15344). In paraffin-section IHC, expect cytoplasmic staining in selected glandular cells, kidney tubule cells and glial cells (HPA tissue IHC). HPA rates its tissue staining Approved but reports low consistency with RNA expression, so use the pattern as an interpretation guide rather than proof of target identity (HPA tissue IHC).

What am I looking at on my slide?
Moderate cytoplasmic staining in colon or duodenal glandular cells, kidney tubule cells, or glial cells in cerebral cortex (HPA tissue IHC).	This fits the reported cell types, intensity and compartment (HPA tissue IHC). Compare staining with adjacent cells and a counterstain before scoring; a positive field need not mean every cell is positive (standard IHC practice).
Strong nuclear-only, membrane-rim or extracellular staining dominates the section.	Those patterns conflict with the reported cytoplasmic distribution and absence of a transmembrane segment (HPA tissue IHC; UniProt O15344). Treat them as suspect and check the negative reagent control and staining conditions before assigning MID1 positivity (standard IHC practice).
Adipocytes or bone-marrow hematopoietic cells stain as strongly as the expected positive cells.	HPA reports MID1 as not detected in those cell populations (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity; compare a matched negative control before attributing the signal to MID1 (standard IHC practice).
Brown precipitate covers many compartments or obscures cell boundaries.	Diffuse background prevents a reliable cytoplasmic call (standard IHC practice). Review reagent controls, blocking, washes and detection exposure; reduce background before comparing intensity across cell types (standard IHC practice).
No staining appears in a section containing an expected positive cell population.	First confirm that the relevant cells are present: HPA reports medium staining in colon glands and kidney tubules, not uniform staining across either tissue (HPA tissue IHC). Then inspect controls and the validated IHC conditions; HPA's low RNA–staining consistency limits biological conclusions from a single negative slide (HPA tissue IHC; standard IHC practice).

💡Expected MID1 appearanceCall a result consistent with MID1 when medium cytoplasmic staining appears in the specified glandular, kidney tubule or glial cells (HPA tissue IHC); dominant nuclear, membrane or extracellular signal is suspect (HPA tissue IHC; UniProt O15344).

How each factor affects the staining
Cell type and comparator tissue	HPA reports medium staining in selected appendix, colon, duodenum and endometrium glandular cells, kidney tubules and brain glial cells; adipocytes are not detected (HPA tissue IHC). Score identified cells, not the whole section, and use the latter pattern as a comparator with appropriate controls (standard IHC practice).
Compartment and cell-cycle context	MID1 associates with cytoplasmic microtubules in interphase and with the mitotic spindle and midbodies during division (UniProt O15344). HPA's tissue-level description is cytoplasmic; routine chromogenic IHC need not resolve those fine structures (HPA tissue IHC; standard IHC practice).
Antibody validation and evidence limit	HPA003715 is Approved for IHC, while HPA002878 has no listed IHC validation; HPA also reports low consistency between staining and RNA expression (HPA antibodies; HPA tissue IHC). Match any interpretation to the antibody actually used, and avoid treating an Approved label alone as independent confirmation of specificity.
Protein forms	UniProt lists two MID1 isoforms and a full-length chain spanning residues 1–667, with no signal peptide or propeptide (UniProt O15344). The supplied sources give no antibody epitope or isoform-specific IHC pattern, so they cannot justify different staining calls for the two isoforms.
IF/ICC cross-check	HPA reports supported cytosolic localization in ICC-IF, with centriolar satellite and Golgi assignments marked uncertain (HPA subcellular). These observations may inform interpretation, but they do not establish that fine puncta should be visible in paraffin-section chromogenic IHC (HPA subcellular; standard IHC practice).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected positive cells are present, but the IHC slide is blank.	The result may reflect an unsuccessful staining run or a weak sample signal; HPA reports medium staining in selected cells, with low RNA–staining consistency (HPA tissue IHC; standard IHC practice).	Check the run control, antibody identity and the catalog antibody's IHC-P conditions, including its stated retrieval and dilution if supplied; do not infer a MID1-specific retrieval or fixation effect from these sources (standard IHC practice).
The entire section has widespread cytoplasmic color.	The distribution is broader than HPA's cell-specific examples, and diffuse background can obscure selective staining (HPA tissue IHC; standard IHC practice).	Inspect a negative reagent control, then review blocking, washes and detection exposure before rescoring identified cells (standard IHC practice).
Color is concentrated in nuclei or along cell borders.	Dominant nuclear or membrane staining does not fit HPA's cytoplasmic profile or UniProt's nonmembrane topology (HPA tissue IHC; UniProt O15344).	Check morphology with the counterstain, compare control sections and avoid scoring the misplaced signal as confirmed MID1 (standard IHC practice).
An HPA-listed negative cell population stains prominently.	HPA reports no detection in adipocytes and bone-marrow hematopoietic cells; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC; standard IHC practice).	Compare the same cell population in a negative reagent control and a reported positive population in the run before interpreting it as MID1 (HPA tissue IHC; standard IHC practice).
Two antibodies give different tissue patterns.	Their listed validation differs: HPA003715 is Approved for IHC, whereas HPA002878 has no listed IHC status; the tissue profile also has low RNA–staining consistency (HPA antibodies; HPA tissue IHC).	Record which antibody produced each pattern and assess each against its own validation and controls; do not assume either pattern is confirmed solely by the other antibody's status (HPA antibodies; standard IHC practice).
Can an ICC-IF punctate pattern settle an ambiguous IHC result?	HPA calls the centriolar satellite and Golgi ICC-IF locations uncertain, while cytosol is supported (HPA subcellular).	Use ICC-IF as a separate localization comparison, then decide the paraffin IHC result from its cytoplasmic cell-type pattern and IHC controls (HPA tissue IHC; standard IHC practice).

Source: UniProt O15344 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing MID1 staining

IHC analysis of MID1 using anti-MID1 antibody (A01774-1). MID1 was detected in paraffin-embedded section of human placenta tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-MID1 Antibody (A01774-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

A01774-1 · IHCIHC analysis of MID1 using anti-MID1 antibody (A01774-1). MID1 was detected in paraffin-embedded section of human placenta tissues. Heat med

IHC analysis of MID1 using anti-MID1 antibody (A01774-1). MID1 was detected in paraffin-embedded section of mouse brain tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-MID1 Antibody (A01774-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

A01774-1 · IHCIHC analysis of MID1 using anti-MID1 antibody (A01774-1). MID1 was detected in paraffin-embedded section of mouse brain tissues. Heat mediat

IF analysis of MID1 using anti-MID1 antibody (A01774-1). MID1 was detected in immunocytochemical section of U20S cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-MID1 Antibody (A01774-1) overnight at 4°C. DyLight®594 Conjugated Goat Anti-Rabbit IgG (BA1142) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A01774-1 · IFIF analysis of MID1 using anti-MID1 antibody (A01774-1). MID1 was detected in immunocytochemical section of U20S cells. Enzyme antigen retri

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000101871-MID1/tissue/cerebral+cortex#imid_1291620

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000101871-MID1/tissue/colon#imid_1291546

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000101871-MID1/tissue/liver#imid_1291547

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000101871-MID1/tissue/kidney#imid_1291548

HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA002878; green = MID1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for MID1 IHC & IF

🧪Run appendix first: glandular cells should show medium MID1 staining (HPA: Appendix glandular cells, Medium). Use adipose tissue adipocytes as a negative comparator (HPA: Not detected); on the appendix slide, treat non-glandular cells as internal background comparators, since their MID1 status is not supplied (HPA: Appendix glandular-cell row only).

Positive control tissue: Appendix (Glandular cells, HPA Medium)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show MID1 in A-549, GAMG, U2OS, with annotated localisation: Centriolar satellite (uncertain) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control, a concentration-matched nonimmune rabbit IgG isotype control, and a matched MID1 knockout biological control if available (caption: rabbit anti-MID1 primary; standard IHC practice). Quench endogenous peroxidase and check for endogenous biotin background in appendix when using the reported biotin/SABC-DAB detection (caption: biotinylated secondary and SABC-DAB; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A01774-1 paraffin-section caption does not state the fixative (caption: fixative not stated). Heat-mediated citrate retrieval at pH 6 for 20 minutes was used for the placenta image; whether appendix staining depends on those conditions is unreported (caption: placenta IHC). IF may help assess MID1's microtubule-associated cytoplasmic localization (UniProt O15344: subcellular location), but the supplied evidence does not establish that IF or frozen sections are easier, or document an appendix-specific artefact.

HPA tissue IHC evidence for MID1

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Positive expression · recommended positive controls

Tissue	Cell type	Level	Evidence	Source
Appendix	Glandular cells	Medium	Protein (IHC)	HPA →
Caudate	Glial cells	Medium	Protein (IHC)	HPA →
Cerebral cortex	Glial cells	Medium	Protein (IHC)	HPA →
Colon	Glandular cells	Medium	Protein (IHC)	HPA →
Duodenum	Glandular cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Cervix	Glandular cells	Not detected	Protein (IHC)	HPA →
Esophagus	Squamous epithelial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced MID1 IHC Tips

Troubleshoot MID1 staining in paraffin sections by checking retrieval, cytoplasmic localisation, cell identity and controls before interpreting signal intensity.

What retrieval should I try first when MID1 staining is weak?

Start with heat-mediated citrate buffer at pH 6 for 20 minutes on paraffin sections (datasheet A01774-1). The catalog antibody’s placenta image used that retrieval, followed by 10% goat serum blocking and 1 μg/mL primary antibody overnight at 4°C (IHC caption A01774-1). If staining remains weak, compare retrieval time on adjacent sections while keeping detection and imaging conditions matched (standard IHC practice). Include a no-primary control to distinguish improved MID1 signal from retrieval-related background, and assess cytoplasmic staining in the same cell populations across conditions (UniProt O15344 localisation; standard IHC practice).

How should I troubleshoot inconsistent MID1 staining across paraffin blocks?

Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (IHC caption A01774-1). Record each block’s fixation history and compare similarly processed sections before attributing staining differences to MID1 abundance (standard IHC practice). Hold the documented citrate pH 6, 20-minute retrieval and 1 μg/mL overnight primary incubation constant while assessing block-to-block variation (IHC caption A01774-1). Check tissue preservation and whether staining follows comparable cell compartments; damaged morphology or uneven staining warrants caution in scoring (standard IHC practice). Neither tissue staining patterns nor MID1’s cytoplasmic location establishes a fixation effect (HPA tissue IHC; UniProt O15344 localisation).

Where should convincing MID1 chromogenic staining appear?

Assess MID1 primarily as a cytoplasmic signal, since it associates with microtubules and can localise to spindle and midbody structures during division (UniProt O15344 localisation). HPA tissue IHC likewise describes cytoplasmic expression across several tissues, although its antibody staining has low consistency with RNA expression (HPA tissue IHC). In the selected placenta paraffin-section image, evaluate DAB within intact cells against nearby unstained areas and a no-primary control (IHC caption A01774-1; standard IHC practice). Strong nuclear-only or membranous staining warrants a specificity check because MID1 has no transmembrane segment and its recorded locations are cytoplasmic (UniProt O15344 topology and localisation).

Could isoforms or epitope accessibility explain discordant MID1 staining?

MID1 has 2 recorded isoforms, so an antibody’s recognised epitope matters when comparing staining across samples (UniProt O15344 isoforms; standard IHC practice). Check the catalog antibody’s stated immunogen or epitope against each isoform before interpreting a negative section as absent MID1 (standard IHC practice). The record lists phosphoserines at positions 92, 96 and 511, but does not establish that these modifications alter this antibody’s staining (UniProt O15344 modified residues). Compare sections under the same citrate pH 6, 20-minute retrieval and use matched positive and no-primary controls to separate variable staining from epitope uncertainty (IHC caption A01774-1; standard IHC practice).

How can IF help check an ambiguous MID1 IHC pattern?

Use IF as a complementary localisation check and consult its separate guide for application-specific validation (standard IF practice). Multiplex MID1 with a marker identifying the cell population under study; HPA reports medium IHC staining in kidney tubule cells and several glandular or glial populations, with low antibody–RNA consistency (HPA tissue IHC). Choose spectrally separated fluorophores and inspect unstained tissue for autofluorescence before assigning a faint signal to MID1 (standard IF practice). Because MID1 is cytoplasmic and has no transmembrane segment, assess permeabilisation for access to an intracellular epitope (UniProt O15344 topology and localisation; standard IF practice). Compare cellular patterns with the paraffin-section DAB result without treating them as interchangeable validation (standard IHC/IF practice).

What should I change when MID1 DAB staining is diffuse?

First compare the stained section with a no-primary control and inspect section edges and damaged regions for nonspecific deposition (standard IHC practice). The selected paraffin-section image used 10% goat serum blocking, 1 μg/mL primary overnight at 4°C, and biotinylated secondary followed by a streptavidin–biotin complex and DAB (IHC caption A01774-1). If background persists, titrate primary antibody and review blocking, washes, endogenous peroxidase blocking and biotin-related background in this detection workflow (standard IHC practice). Preserve the documented citrate pH 6 retrieval while testing one change at a time, then judge whether intact cells retain a plausible cytoplasmic pattern (IHC caption A01774-1; UniProt O15344 localisation; standard IHC practice).

How should I score heterogeneous MID1 staining in paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define cell populations and regions before scoring, then record both the percentage of positive cells and staining intensity within each population (standard IHC practice). An H-score can combine intensity categories with their percentages; alternatively, report positive-cell density per mm² when cell counts and sampled area are reliable (standard IHC practice). Normalise to the number of evaluable target cells or tissue area, and keep section processing, DAB development and thresholds consistent across cases (standard IHC practice). Report glandular, glial or tubule populations separately where relevant, since HPA describes differing cell-specific staining and low consistency between antibody staining and RNA data (HPA tissue IHC). Exclude folds, edges and necrotic areas before calculating scores (standard IHC practice).

When is a MID1-positive IHC result credible?

Prioritise staining within intact cell cytoplasm, consistent with MID1’s microtubule-associated localisation and HPA’s tissue IHC profile (UniProt O15344 localisation; HPA tissue IHC). Check the identity of stained cells: HPA reports medium staining in kidney tubule cells and some glandular and glial populations, but its staining–RNA consistency is low (HPA tissue IHC). Treat nuclear-only, surface-like, edge-restricted or necrotic staining cautiously, and compare no-primary and endogenous-peroxidase controls before assigning DAB signal to MID1 (UniProt O15344 topology and localisation; standard IHC practice). The selected placenta image demonstrates staining with this catalog antibody in paraffin sections, while its caption leaves the fixative unspecified (IHC caption A01774-1).

Boster reagents

Best MID1 / E3 ubiquitin-protein ligase MID1 IHC Antibodies

A01774-1 has IHC images from paraffin sections of human placenta and mouse brain (catalog IHC captions), plus IF/ICC data from U20S cells (catalog IF caption).

Real IHC data

Anti-MID1 Antibody ®

Cat # A01774-1

View product →

A01774-1 is listed for IHC in human and mouse (catalog: applications and reactivity), with paraffin-section images from human placenta and mouse brain (catalog IHC captions). It is also listed for IF/ICC (catalog: applications), with a cell image labeled U20S (catalog IF caption).

Which to pick: Choose A01774-1 for paraffin-section IHC; its own captions show citrate retrieval and 1 μg/ml antibody on human placenta and mouse brain (catalog IHC captions). The same SKU covers IF/ICC in U20S cells at 2 μg/ml (catalog IF caption) and is listed as human- and mouse-reactive (catalog: reactivity). Its host is rabbit, while clonality and the IHC tissue fixative are unreported (catalog: host and clone; catalog IHC captions).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry O15344 (TRI18_HUMAN, E3 ubiquitin-protein ligase MID1).
Human Protein Atlas. MID1 tissue IHC expression (reliability: Approved).
Human Protein Atlas. MID1 subcellular location (ICC-IF): Mainly localized to the centriolar satellites. In addition localized to the Golgi apparatus and cytosol..
Human Protein Atlas. MID1 antibody validation summary (2 antibodies).
Resveratrol induces dephosphorylation of Tau by interfering with the MID1-PP2A complex. Scientific reports 2017 — PMC5653760.
A hormone-dependent feedback-loop controls androgen receptor levels by limiting MID1, a novel translation enhancer and promoter of oncogenic signaling. Molecular cancer 2014 — PMC4074869.
Midline-1 inhibited high glucose-induced epithelial-mesenchymal transition, fibrosis and inflammation through WNT/β-catenin signaling in benign prostatic hyperplasia. Frontiers in endocrinology 2025 — PMC11978649.
Exosomes derived from myeloid-derived suppressor cells facilitate castration-resistant prostate cancer progression via S100A9/circMID1/miR-506-3p/MID1. Journal of translational medicine 2022 — PMC9344715.
PubMed PMID:9354791 — UniProt-cited evidence.
PubMed PMID:9425238 — UniProt-cited evidence.
PubMed PMID:9722948 — UniProt-cited evidence.