# Indirect Fluorescent Antibody Test: How It Works
The indirect fluorescent antibody test, usually shortened to IFA or IIF, detects antibodies in a patient sample by using a labeled secondary reagent instead of labeling the patient's own antibody. The test answers a single question: does this serum contain antibodies that bind a specific antigen, and if so, how much? It is the backbone of autoimmune serology (antinuclear antibodies on HEp-2 cells), infectious disease serology (Toxoplasma, Brucella, hepatitis E), and veterinary antibody screening. It is also the method behind the Crithidia luciliae indirect immunofluorescence test for anti-dsDNA antibodies in lupus [1].
The method works because two binding events stack on top of each other. Patient antibodies bind the fixed antigen on a slide. Then a fluorochrome-tagged anti-species antibody binds those patient antibodies. The second step is why the assay is called indirect: the fluorescent signal is generated by a reagent that recognizes the patient's antibody, not by the patient's antibody itself. That design gives IFA two advantages over direct fluorescence: one labeled reagent can detect antibodies from many patients, and the layered binding amplifies signal.
Hands-on time for a typical IFA run is roughly 60 to 90 minutes from slide retrieval to the first microscopic read, with about 30 to 45 minutes of that being active pipetting and washing. Elapsed time including incubations and mounting is usually 2 to 3 hours. Batch size drives throughput: a single technologist can process 20 to 40 slides in one run, but each slide must be read manually under a fluorescence microscope, which is the true bottleneck.
This guide walks through the full workflow, the control setup that keeps results honest, and how IFA compares with ELISA and direct fluorescent antibody testing.
## What IFA Detects and When to Use It
IFA detects antibodies, not antigens. That distinction matters for interpretation. A positive IFA tells you the patient has been exposed to an antigen or has an autoantibody, not that the organism is currently replicating in tissue. For acute infection, paired sera showing a rising titer or a positive IgM-class result carries more weight than a single IgG titer.
Use IFA when you need to detect antibodies against a defined antigen that can be fixed to a slide, when you want to see the spatial pattern of binding (nuclear, cytoplasmic, membrane, or organism-specific), or when no reliable ELISA exists for the target. The pattern information is a genuine advantage. In antinuclear antibody testing, the staining pattern on HEp-2 cells (homogeneous, speckled, nucleolar, centromeric) guides which specific antigen tests to order next, and laboratories report both titer and pattern for this reason [2].
IFA is also used when a solid-phase assay gives an ambiguous result and a second method is needed. In hepatitis E virus serology, an immunofluorescence test based on mammalian cells expressing recombinant ORF2 protein confirmed infections and supported diagnosis when commercial assays gave ambiguous results [3]. The same logic applies in anti-dsDNA testing, where the Crithidia luciliae IFA is used as a highly specific confirmatory method alongside sensitive solid-phase immunoassays [1].
IFA is not always the right first choice. It is labor-intensive, requires a fluorescence microscope and a trained reader, and is harder to standardize across laboratories than plate-based assays. A national survey of Spanish autoimmunity laboratories found that 84% used HEp-2 IIF as the screening method for antinuclear antibodies, but testing practices were highly heterogeneous for items such as serum dilutions and the minimum interval for repeating tests [2]. That heterogeneity is a real limitation of the format, not a reason to abandon it.
## Principle of the Assay
Indirect immunofluorescence micrograph: fluorescent antibody staining reveals target organisms against a dark background. Image: Tobili Sam-Yellowe, Raghavendra Yadavalli, CC BY-SA 4.0, via Wikimedia Commons.
The indirect fluorescent antibody test is a sandwich assay built on a solid phase.
1. Antigen is fixed to a glass slide well. The antigen can be whole organisms (Toxoplasma tachyzoites, Brucella), cultured cells (HEp-2, Vero), tissue sections, or transfected cells expressing a recombinant protein.
2. Patient serum is diluted and applied. Any antibodies specific for the fixed antigen bind and stay bound after washing.
3. A fluorochrome-conjugated secondary antibody is applied. This reagent is raised against the immunoglobulin of the patient's species (anti-human IgG, anti-canine IgG, anti-mouse IgG) and carries a fluorescent dye such as fluorescein isothiocyanate (FITC) or a DyLight dye.
4. After a second wash, the slide is mounted and read under a fluorescence microscope. Bright apple-green (FITC) or red (DyLight 488 is read in the green channel, Texas Red in the red channel) fluorescence at the location of the antigen indicates a positive result.
The signal is proportional to the amount of patient antibody bound, within limits. That proportionality is the basis for endpoint titration, where the titer is the highest dilution that still gives visible specific fluorescence.
### Why the Secondary Antibody Must Match the Species
The conjugate is species-specific. An anti-human IgG conjugate will not detect dog, horse, or mouse antibodies, and an anti-mouse conjugate will not detect human antibodies. This is the single most common setup error in mixed-species work. If you are testing primate sera with a human conjugate, verify cross-reactivity before reporting results, because anti-human reagents vary in how well they recognize non-human primate immunoglobulin. In one comparison of IFA and the modified agglutination test for anti-Toxoplasma IgG in Neotropical primates, agreement between the two methods was low (Kappa = 0.21), with only 3 animals positive by IFA versus 20 by MAT [4]. Species mismatch or poor conjugate performance is one plausible contributor to that kind of discordance, and it is a reason to validate any cross-species application locally rather than assuming a human kit will work.
Some newer formats sidestep species specificity entirely. A point-of-care Brucella assay used protein A conjugated to aggregation-induced emission fluorescent microspheres as a universal detection probe, recognizing Brucella antibodies across multiple species without species-specific secondary antibodies [5]. That is a design solution to the species problem, not a property of conventional IFA.
## Materials and Reagents
Working concentrations vary by manufacturer and target. The table below gives typical working ranges for conventional IFA and the role of each reagent. Always follow the validated package insert for a specific kit, because fixation, blocking, and conjugate dilution are kit-specific.
| Reagent | Typical working concentration or format | Role |
|--|--|--|
| Antigen slides | Pre-coated wells, air-dried, fixed | Solid phase presenting the target antigen |
| Patient serum | Screen at 1:20 to 1:40, then two-fold dilutions to endpoint | Source of test antibody |
| PBS, pH 7.2 to 7.4 | 1x working strength | Diluent and wash buffer |
| Blocking reagent | 1% to 5% bovine serum albumin or normal goat serum in PBS | Reduces nonspecific conjugate binding |
| Fluorochrome-conjugated secondary antibody | Use at the titer stated on the insert, commonly 1:50 to 1:200 | Detects bound patient antibody |
| Evans blue counterstain | 0.005% to 0.01% in mounting medium or conjugate diluent | Quenches background and improves contrast |
| Mounting medium | Glycerol-based, pH 8.0 to 9.0, with antifade | Preserves fluorescence and covers the well |
| Known positive control serum | Defined titer, species-matched | Confirms antigen and conjugate performance |
| Known negative control serum | Species-matched, antibody-negative | Confirms specificity and low background |
| Conjugate-only control | PBS substituted for patient serum | Detects conjugate binding to antigen or slide |
### Safety Note
Sodium azide is a common preservative in conjugate buffers and is toxic if ingested and reactive with copper and lead plumbing. Do not pour azide-containing waste down a sink. Handle patient sera as potentially infectious and process them in a biological safety cabinet when aerosol generation is possible. Fluorescence microscope lamps, particularly mercury vapor sources, emit ultraviolet light that can damage eyes, so use the appropriate shield and never look directly at an operating lamp.
## Step-by-Step Procedure
Each step below includes the reason it matters. Skipping or shortening a wash is the most common cause of a false positive.
1. **Equilibrate slides and reagents to room temperature.** Cold slides condense moisture and dilute reagents unevenly. Bring slides to room temperature for 10 to 20 minutes before opening the pouch.
2. **Label slides and mark wells.** Use a diamond pencil or a permanent marker on the frosted end. Label each well with the sample identity and dilution. Mislabelled wells are unrecoverable.
3. **Prepare serum dilutions in PBS.** Start with a screening dilution, commonly 1:20 or 1:40, then prepare two-fold serial dilutions (1:40, 1:80, 1:160, and so on) for endpoint titration. Change pipette tips between dilutions to avoid carryover.
4. **Apply diluted serum to wells.** Use enough volume to cover the well completely, typically 10 to 25 microliters depending on well size. Underfilling leaves antigen unreacted and produces patchy staining.
5. **Incubate in a humid chamber at 37 degrees Celsius for 30 minutes.** Humidity prevents evaporation, which concentrates salts and causes artifactual bright edges. Do not let wells dry at any point in the assay.
6. **Wash three times with PBS.** Use a gentle stream and a 5-minute soak for at least one wash. Washing removes unbound antibody. Insufficient washing leaves unbound patient antibody that the conjugate will detect, creating a false positive.
7. **Apply the fluorochrome-conjugated secondary antibody.** Use the working dilution from the insert. The conjugate must be specific for the patient's species and, if you need class-specific information, for the immunoglobulin class (anti-IgG versus anti-IgM).
8. **Incubate in a humid chamber at 37 degrees Celsius for 30 minutes, protected from light.** Fluorochromes photobleach. Keep the chamber covered or work under subdued light.
9. **Wash three times with PBS as before.** A second thorough wash removes unbound conjugate, which is the main source of diffuse background fluorescence.
10. **Apply mounting medium and a coverslip.** Mounting medium with an antifade agent and a pH of 8.0 to 9.0 preserves FITC fluorescence, which is pH-sensitive and dims below pH 7.
11. **Read under a fluorescence microscope.** Use the filter cube matched to the fluorochrome (blue excitation for FITC, green excitation for Texas Red). Start with the known positive control to confirm the system is working, then read negative controls, then patient wells.
12. **Record titer and pattern.** Report the highest dilution with specific fluorescence and describe the pattern (nuclear, cytoplasmic, membrane, organism-shaped). Pattern is diagnostic information, not decoration.
### Workflow Overview
The diagram below shows the decision path from slide setup through reporting.
```mermaid
flowchart TD
A[Retrieve antigen slides] --> B[Apply diluted patient serum]
B --> C[Incubate 37 C 30 min]
C --> D[Wash three times]
D --> E[Apply fluorescent conjugate]
E --> F[Incubate 37 C 30 min dark]
F --> G[Wash three times]
G --> H[Mount and coverslip]
H --> I[Read positive control]
I --> J{Positive control bright}
J -->|No| K[Troubleshoot reagent or microscope]
J -->|Yes| L[Read negative and conjugate controls]
L --> M[Read patient wells and record titer]
```
## Controls: The Part That Makes Results Trustworthy
Every IFA run needs three controls. Without them, a result is an observation, not a measurement.
**Known positive control.** A species-matched serum with a defined antibody titer. It confirms that the antigen is present and reactive, the conjugate is active, and the microscope and filter are working. If the positive control is dim or negative, the entire run is invalid regardless of what the patient wells show.
**Known negative control.** A species-matched serum that is antibody-negative for the target. It confirms that the conjugate is not binding nonspecifically to the antigen or the slide and that background is acceptably low. A bright negative control means the conjugate concentration is too high, blocking is inadequate, or the wash was insufficient.
**Conjugate-only control.** PBS or diluent replaces the patient serum, and the conjugate is applied as usual. This well isolates conjugate behavior. If it fluoresces, the conjugate is binding directly to the antigen or the slide surface, and every patient result is suspect. Conjugate-only controls are especially important when you switch antigen lots, conjugate lots, or blocking reagents.
Run all three controls on every slide or at minimum on every run, depending on your laboratory's validation. The conjugate-only control is the one most often omitted and the one that catches the most misleading results.
### Expected Results and How to Read Them
| Control or sample | Expected result | Interpretation if abnormal |
|--|--|--|
| Known positive | Specific fluorescence at the antigen location, at or near the stated titer | Dim or absent: antigen degraded, conjugate inactive, or microscope fault |
| Known negative | No specific fluorescence, low background | Bright: nonspecific binding, conjugate too concentrated, or poor washing |
| Conjugate-only | No specific fluorescence | Bright: conjugate binds antigen or slide directly, results invalid |
| Patient positive | Specific fluorescence that dilutes out in a titer pattern | Bright at all dilutions: probable nonspecific binding or very high titer, retest |
| Patient negative | No specific fluorescence at screening dilution | Faint specific signal: repeat with a lower starting dilution |
Endpoint titer is the reciprocal of the highest dilution showing specific fluorescence. A titer of 1:160 means the 1:160 well was the last positive well. Titers are read as two-fold steps, so a one-step change (1:80 to 1:160) is within assay noise and should not be over-interpreted as a clinical change.
## IFA Compared With ELISA and Direct FA
The three formats answer related questions with different tradeoffs. No single format wins on every axis.
| Feature | Indirect FA (IFA) | ELISA | Direct FA (DFA) |
|--|--|--|--|
| What it detects | Antibodies in patient serum | Antibodies or antigens, depending on format | Antigen in patient specimen |
| Signal amplification | Two-layer binding amplifies signal | Enzymatic amplification, often more sensitive than IFA | Single-layer, generally less sensitive than IFA |
| Typical sensitivity | Moderate to high, antigen-dependent | High, especially optimized indirect ELISA | Moderate, depends on antigen load |
| Equipment needed | Fluorescence microscope, humid chamber, dark storage | Plate reader, washer, incubator | Fluorescence microscope |
| Throughput | Low to moderate, manual reading is the bottleneck | High, 96-well plates read in minutes | Low to moderate, manual reading |
| Quantitative output | Semi-quantitative, endpoint titer | Quantitative or semi-quantitative optical density | Qualitative, presence or absence |
| Pattern information | Yes, spatial pattern is visible | No | Yes, organism morphology is visible |
| Species-specific conjugate | Required | Required for indirect formats | Required |
| Best use case | Autoantibody screening, confirmatory serology, pattern-based diagnosis | High-volume antibody screening, quantitative titering | Direct antigen detection in clinical specimens |
A few points deserve emphasis. An indirect ELISA developed for avian leukosis virus antibodies showed higher agreement than the fluorescent antibody test and was more sensitive and specific than a commercial kit when FAT was the reference method [6]. A multi-peptide indirect ELISA for Senecavirus A antibodies showed no cross-reactivity with other common swine viruses and correlated strongly with virus neutralization, with an AUC of 0.955 against IFA [7]. These are examples of ELISA outperforming IFA on sensitivity and throughput for specific targets. They are not evidence that ELISA replaces IFA universally. IFA retains advantages in pattern recognition, in confirmatory roles where specificity matters more than sensitivity, and in targets where no validated ELISA exists.
Direct FA is a different assay. It uses a fluorochrome-labeled antibody applied directly to a specimen to detect antigen, such as Giardia cysts and Cryptosporidium oocysts in stool [8]. DFA skips the patient-antibody step entirely, so it detects current antigen presence rather than immune response. A multiplex fluorescent antibody microscopy test using DyLight-488-labeled antibodies detected Giardia and Cryptosporidium directly in stool samples with 88.0% sensitivity and 100% specificity for Giardia relative to [multiplex PCR](/knowledge/diagnostics/molecular/multiplex-pcr-design-optimization-and-troubleshooting) [8]. DFA is faster and simpler than IFA but less sensitive because there is no signal amplification layer.
## Common Mistakes and Limitations
**Autofluorescence.** Some antigens, fixatives, and mounting media fluoresce on their own in the FITC channel. Connective tissue, plant material, and certain cell lines are common offenders. Autofluorescence appears as diffuse, often yellow-green or orange, background that does not dilute out with serum titer. Confirm it by reading an unstained well or a well with no conjugate. Evans blue counterstain and narrow-band filter cubes reduce the problem but do not eliminate it.
**Cross-reactivity.** Antibodies can bind antigens that are structurally similar to the target. This produces a positive result in a patient who was never exposed to the specific organism. Cross-reactivity is a particular concern with closely related organisms and with autoimmune sera that contain multiple autoantibodies. Always interpret IFA results in clinical context and confirm unexpected positives with an orthogonal method.
**Species-specific conjugate mismatch.** Using an anti-human conjugate on non-human serum, or vice versa, produces false negatives or weak, uninterpretable results. Verify conjugate specificity against the actual patient species before reporting.
**Titer interpretation errors.** A single titer is a snapshot. A titer of 1:160 in a healthy animal and 1:160 in a sick animal mean different things. Paired sera collected 2 to 4 weeks apart showing a four-fold rise are far more informative than any single value. Do not treat a low positive titer as diagnostic on its own, and do not treat a negative result as ruling out exposure if the sample was collected too early in the immune response.
**Wash and drying errors.** Drying wells during the assay concentrates salts and produces bright artifactual edges. Insufficient washing leaves unbound antibody or conjugate and produces false positives. Both are preventable with a humid chamber and a timed wash protocol.
**Photobleaching.** FITC fades under prolonged illumination. Read slides promptly after mounting, and store mounted slides in the dark at 2 to 8 degrees Celsius if they must be held. Re-reading a slide hours later can produce a falsely weaker result.
**Standardization drift.** The same sample can give different titers across laboratories because of differences in antigen preparation, conjugate dilution, and reading thresholds. A study of ANCA indirect immunofluorescence found that protocol modifications significantly increased the positivity rate (14.8% versus 34.3%) and median titers in patients with ANCA-associated vasculitis, autoimmune hepatitis, and ulcerative colitis [9]. That is a caution about comparing results across sites without harmonization.
**Serum storage effects.** IFA titers are relatively robust to short-term temperature excursions. In a study of pemphigus and bullous pemphigoid sera stored at 24 or 40 degrees Celsius for 7 days, 95% and 76% of pemphigus specimens and 89% and 82% of bullous pemphigoid specimens showed no titer difference compared with -80 degrees Celsius controls, and no specimen converted from positive to negative [10]. This does not mean storage conditions are irrelevant. It means a single moderate excursion does not invalidate a result, while prolonged improper storage still should be avoided.
## Variations on the Format
**Class-specific conjugates.** Anti-IgG and anti-IgM conjugates distinguish recent from past infection. IgM appears earlier and wanes faster. A hepatitis E immunofluorescence test detected anti-HEV IgG and IgA but not IgM, illustrating that conjugate choice determines what the assay can and cannot see [3].
**Multiplex and bead-based formats.** Magnetic protein microbead-aided indirect fluoroimmunoassays use an avidin-biotin system to attach viral antigens to beads and detect antibodies with FITC-labeled secondary reagents [11]. These formats allow higher throughput and automation than slide-based IFA.
**Near-infrared and alternative fluorophores.** Research applications use near-infrared dyes and antibody-fluorophore conjugates for in vivo imaging, where tumor-to-background ratios and dye-to-protein ratios are optimized [12]. These are not diagnostic IFA formats, but they demonstrate the same antibody-conjugate chemistry at a different scale.
**Sequential staining and elution.** Indirect immunofluorescence is usually limited to 3 to 5 markers per preparation. A 2-mercaptoethanol and SDS solution can elute primary and secondary antibody layers and allow sequential staining, though biotin-streptavidin bonds resist elution [13]. This is a research technique, not a routine diagnostic method.
## Storage and Stability Notes
Store antigen slides sealed with desiccant at 2 to 8 degrees Celsius and use before the expiry date. Do not freeze slides, because freeze-thaw cycles crack the antigen coating. Bring slides to room temperature before opening the pouch to prevent condensation.
Store fluorochrome-conjugated antibodies at 2 to 8 degrees Celsius, protected from light, at the concentration supplied. Avoid repeated freeze-thaw cycles if the conjugate is supplied frozen. Diluted conjugate should be used the same day.
Store patient sera at 2 to 8 degrees Celsius for testing within a few days, or at -20 to -80 degrees Celsius for longer storage. The pemphigus and bullous pemphigoid data suggest short-term room-temperature exposure does not destroy IFA reactivity, but validated cold storage remains the standard [10].
Store mounting medium at room temperature, tightly capped, and discard if it becomes cloudy or if the pH drifts, because FITC fluorescence depends on an alkaline pH.
## Frequently Asked Questions
### What is the difference between indirect and direct fluorescent antibody testing?
Indirect FA detects antibodies in patient serum using a labeled secondary antibody, while direct FA detects antigen in a specimen using a labeled primary antibody. Indirect FA has an extra amplification step and gives titer and pattern information. Direct FA is faster and detects current antigen presence.
### Why does the conjugate have to be species-specific?
The conjugate recognizes the constant region of the patient's immunoglobulin. An anti-human conjugate will not bind dog or mouse antibodies, so a mismatch produces false negatives or weak results. Always match the conjugate to the species being tested.
### What controls are required for a valid IFA run?
A known positive control, a known negative control, and a conjugate-only control. The positive control confirms the system works, the negative control confirms specificity, and the conjugate-only control detects direct conjugate binding that would invalidate patient results.
### Can IFA results be compared between laboratories?
Only with caution. Antigen preparation, conjugate dilution, and reading thresholds vary, and protocol differences can shift positivity rates and titers substantially. Use the same laboratory for serial samples whenever possible.
### What does a titer mean?
The titer is the reciprocal of the highest dilution that still shows specific fluorescence. A single titer is a snapshot. Paired sera showing a four-fold rise are much more informative than one value.
### What causes a false positive IFA?
Insufficient washing, excessive conjugate concentration, cross-reactive antibodies, and autofluorescence are the main causes. A conjugate-only control and a known negative control will flag most of these problems.
### How should IFA slides be stored?
Store coated slides sealed with desiccant at 2 to 8 degrees Celsius and never freeze them. Store conjugates refrigerated and protected from light. Read mounted slides promptly because fluorochromes photobleach.
### Is IFA better than ELISA?
Neither is universally better. ELISA offers higher throughput and often higher sensitivity for high-volume screening. IFA offers pattern information and a confirmatory role where specificity matters. The right choice depends on the target, the clinical question, and the laboratory's equipment.
## Related Articles
- [Western Blot Test: How It Works and What It Detects](/knowledge/molecular-biology/western-blot-test)
- [Direct vs Indirect Immunofluorescence in Viral Diagnostics](/knowledge/diagnostics/microbiology/direct-vs-indirect-immunofluorescence-in-viral-diagnostics)
- [Heartworm Antigen vs Antibody Tests: Feline vs Canine Diagnosis Protocol](/knowledge/veterinary-medicine/at-home-diagnostics/heartworm-antigen-vs-antibody-tests-feline-vs-canine-diagnosis-protocol)
- [Choosing Fluorescent Dyes and Probes for Multiplex qPCR](/knowledge/molecular-biology/choosing-fluorescent-dyes-and-probes-for-multiplex-qpcr-a-comparison-of-fam-hex-cy5-and-beyond)
- [Antibody Structure](/blog/guides/antibody-structure)
- [Antibody Sequence Database](/blog/guides/antibody-sequence-database)
## Sources
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2. [Laboratory and clinical practices in antinuclear antibody detection and related antigens: recommendations from a Spanish multicentre survey.](https://pubmed.ncbi.nlm.nih.gov/37195550/)
3. [A novel indirect immunofluorescence test for the detection of IgG and IgA antibodies for diagnosis of Hepatitis E Virus infections.](https://pubmed.ncbi.nlm.nih.gov/23557668/)
4. [Comparison Between Indirect Fluorescent Antibody Test and Modified Agglutination Test for Detecting Anti-Toxoplasma gondii IgG Antibodies in Neotropical Primates.](https://pubmed.ncbi.nlm.nih.gov/39834083/)
5. [BEACON: AIE-based point-of-care test for cross-species Brucella antibody detection.](https://pubmed.ncbi.nlm.nih.gov/42296868/)
6. [Development and validation of an indirect enzyme-linked immunosorbent assay for the detection of Avian leukosis virus antibodies based on a recombinant capsid protein.](https://pubmed.ncbi.nlm.nih.gov/21908361/)
7. [Development and application of a novel multi-peptide indirect ELISA based on VP1/VP2/VP3 proteins for the antibody detection against Senecavirus A.](https://pubmed.ncbi.nlm.nih.gov/42061535/)
8. [Diagnostic accuracy of ARK Checker® C/G-DyLight® 488: simultaneous detection of Giardia and Cryptosporidium by fluorescent antibody microscopy.](https://pubmed.ncbi.nlm.nih.gov/41593455/)
9. [Improvement of indirect immunofluorescence technique to detect antineutrophil cytoplasmic antibodies and its impact on test positivity rate.](https://pubmed.ncbi.nlm.nih.gov/37493768/)
10. [Revisiting serum storage protocols: assessing the impact of temperature variations on indirect immunofluorescence testing for pemphigus and bullous pemphigoid.](https://pubmed.ncbi.nlm.nih.gov/39067060/)
11. [Magnetic protein microbead-aided indirect fluoroimmunoassay for the determination of canine virus specific antibodies.](https://pubmed.ncbi.nlm.nih.gov/21300534/)
12. [Fluorescence-Guided Surgery of Rhabdomyosarcoma Using a B7-H3 Targeted Antibody-Fluorophore Conjugate.](https://pubmed.ncbi.nlm.nih.gov/42276839/)
13. [Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus.](https://pubmed.ncbi.nlm.nih.gov/38434276/)
