Comparative Analysis and Selection Strategies for Mounting Media in Immunological Experiments
Comparative Analysis and Selection Strategies for Mounting Media in Immunological Experiments
Mounting is not merely an ancillary step after staining, but a critical determinant of section transparency, fluorescence stability, background control, and storage life. In experiments such as immunofluorescence, immunohistochemistry, in situ hybridization, and lipid staining, different mounting media provide distinct chemical environments, curing behaviors, and sample compatibility profiles. Inappropriate selection of a mounting system is often directly manifested as rapid fluorescence decay, loss of lipid-associated signals, coverslip displacement, increased background, or reduced long-term archival quality. Therefore, the criteria for selecting a mounting medium should not remain at the level of whether a coverslip can simply be applied, but should instead be evaluated comprehensively in relation to the specific experimental type, signal development mode, and preservation objective.
Keywords: mounting medium; antifade; immunofluorescence; immunohistochemistry; glycerol gelatin; PVP; Kisser mounting medium
1 Experimental Role and Classification Basis of Mounting Media
1.1 Position of the Mounting Step in the Experimental Workflow
The direct functions of mounting can be understood at three levels. First, it establishes a stable optical interface, reducing refractive disturbance and local imaging distortion. Second, it limits sample exposure to oxygen, light, and mechanical disturbance, thereby slowing fluorescence decay or deterioration of chromogenic products. Third, it fixes the coverslip in place, facilitating both short-term observation and long-term preservation.
In chromogenic assays, mounting mainly affects transparency, structural clarity, and archival quality. In fluorescence-based assays, mounting is not merely a pre-observation treatment, but directly participates in signal preservation. Particularly in IF and FISH, improper selection of the mounting system can substantially compromise the value of earlier optimization efforts in antibody incubation, staining conditions, and microscopy parameters.
1.2 Major Types of Mounting Media
(1) Antifade mounting media.
These are mainly used in immunofluorescence, FISH, and multiplex fluorescence labeling. Their core function is to reduce photobleaching by means of antioxidants and free-radical scavengers.
(2) Water-soluble mounting media.
These are mainly composed of glycerol, gelatin, PVP, or related components. They do not rely on xylene or dehydration-clearing procedures and are suitable for samples that cannot tolerate organic solvents.
(3) Resin-based mounting media.
Examples include neutral balsam and Canada balsam. These are suitable for non-fluorescent sections that have already undergone dehydration and clearing and can form a relatively stable hard transparent layer after mounting.
Table 1. Basic Classification and Applications of Mounting Systems
Type | Representative Systems | Main Features | Main Applicable Experiments | Main Limitations |
Antifade mounting media | Antifade medium, DAPI-containing antifade medium, PVP-based antifade medium | Protect fluorescence signals and slow photobleaching | IF, FISH, multiplex fluorescence labeling | Not suitable as a substitute for long-term hard resin sealing |
Water-soluble mounting media | Kisser mounting medium, PVP mounting medium, glycerol gelatin | No dehydration or clearing required; relatively mild to samples | Frozen sections, water-soluble stains, some routine sections | Ordinary types usually do not provide antifade capability |
Resin-based mounting media | Neutral balsam, Canada balsam | Dry to form a hard transparent layer suitable for long-term archiving | HE, routine IHC, DAB-developed sections | Organic solvent conditions are unsuitable for fluorescent and lipid-containing samples |
2 Composition Characteristics and Application Boundaries of Common Mounting Media
2.1 Kisser Mounting Medium
(1) Composition and physical characteristics
Kisser mounting medium is a classical water-soluble mounting system, usually based primarily on gelatin and glycerol. It shows clear temperature sensitivity, often appearing gel-like at low temperature or room temperature, while becoming more fluid upon heating, which facilitates dispensing and spreading. After mounting, it usually provides relatively high transparency and stable adhesion and coverage over the tissue surface, making it suitable for routine morphological observation.
(2) Applicable experimental types
Kisser mounting medium is mainly suitable for routine histological mounting in non-fluorescent systems. It performs relatively stably in HE staining, routine IHC, and other sections primarily intended for structural observation. It remains especially practical in experiments requiring a neat overall section appearance, clear tissue boundaries, and relatively long storage periods.
(3) Application advantages
Its main advantages are relatively good transparency and clear tissue contours after mounting, allowing structural layers to be more readily distinguished under a conventional light microscope. Because the sealed state is also relatively stable, it is suitable for teaching slides, routine pathological specimens, and non-fluorescent sections intended for archiving.
(4) Limitations in use
Kisser mounting medium is not an antifade system and is therefore not suitable for experiments such as immunofluorescence that depend on fluorescence signal stability. If a given formulation contains phenol, it may also introduce intrinsic background or interfere with fluorescence observation. Accordingly, in fluorescence experiments, Kisser mounting medium should generally not be selected as a routine mounting option.
2.2 PVP Mounting Medium
(1) Composition and physical characteristics
PVP mounting medium is based on polyvinylpyrrolidone, typically combined with glycerol or similar media to form an aqueous mounting environment. Compared with gelatin-based systems, these media usually show more stable flow behavior and can form a relatively uniform protective film after drying, with less risk of obvious local gel inhomogeneity.
(2) Applicable experimental types
PVP mounting medium is suitable for routine tissue sections, cytology smears, and teaching demonstration samples, especially in high-throughput routine mounting settings. For samples that do not require strong antifade performance but benefit from relatively stable film formation, this system has good operational adaptability.
(3) Application advantages
Its main advantages are ease of use, relatively low volatility, comparatively mild irritancy, and good film-forming properties, so that the coverslip is less prone to obvious shifting after mounting. For routine experiments requiring operational convenience and lower toxic exposure, PVP mounting medium is a practical conventional choice.
(4) Limitations in use
If no antifade component has been incorporated into the PVP system, it is not suitable for samples requiring long-term fluorescence stability. For high-magnification fluorescence imaging or experiments requiring repeated imaging, ordinary PVP mounting medium alone often does not provide sufficient signal protection.
2.3 PVP Antifade Mounting Medium
(1) Composition and physical characteristics
PVP antifade mounting medium is based on a PVP system further supplemented with antioxidant and free-radical scavenging components, thereby combining film-forming protection with fluorescence-preserving properties. After mounting, it can usually provide both relatively good mechanical stability and a certain degree of protection against fluorescence decay.
(2) Applicable experimental types
This system is suitable for immunofluorescence sections, cell coverslips, multiplex fluorescence-labeled samples, and fluorescence experiments requiring transport, re-examination, or short- to medium-term storage. For samples demanding strong coverslip fixation while also requiring antifade protection, this system is usually more practical than a simple liquid antifade medium.
(3) Application advantages
Its advantage lies in combining the mounting stability derived from PVP film formation with the fluorescence-protective capacity of an antifade system. For samples that require repeated observation, repeated photography, or temporary storage before imaging, this type of medium provides a good balance between operational convenience and signal preservation.
(4) Limitations in use
Although suitable for short- to medium-term storage and repeated observation, this type of system is still functionally distinct from traditional resin-based mounting media when the objective is very long-term archiving with a hard-sealed appearance.
2.4 Glycerol Gelatin Mounting Medium
(1) Composition and physical characteristics
Glycerol gelatin consists of gelatin, glycerol, and water and is a typical water-based mounting system. Its key feature is that it does not require dehydration or clearing and is relatively gentle toward lipid-containing components and certain water-soluble chromogenic products.
(2) Applicable experimental types
This system is particularly suitable for Oil Red O lipid staining, frozen sections, certain enzyme histochemistry samples, and experimental materials that should not be exposed to organic solvents. In such specimens, use of clearing systems such as xylene may extract lipids, and some precipitated chromogenic products may also be affected.
(3) Application advantages
The advantage of glycerol gelatin does not lie in having the broadest applicability, but in having a clear and irreplaceable application boundary. In lipid preservation, frozen-section handling, and certain water-soluble chromogenic systems, it can preserve key structures and chromogenic components without damage from organic solvents.
(4) Limitations in use
Ordinary glycerol gelatin does not provide dedicated antifade performance and should therefore not be regarded as a standard mounting system for fluorescence experiments. If the experimental goal is stable preservation of fluorescence signals, dedicated antifade mounting media should still be prioritized.
2.5 Antifade Mounting Media (with or without DAPI)
(1) Composition and physical characteristics
Antifade mounting media are usually based on high-purity glycerol, buffered media, or curable matrices, and are supplemented with antioxidants and free-radical scavengers to reduce photochemical damage to fluorophores during illumination.
(2) Applicable experimental types
These systems are mainly used in IF, FISH, multiplex fluorescent probe experiments, and observation of fluorescent proteins in fixed cells or tissues. Their application range essentially covers all experiments requiring fluorescence microscopy and sensitive to signal decay.
(3) Difference between DAPI-containing and DAPI-free systems
DAPI-free systems are suitable when independent nuclear staining has already been completed and the primary goal is protection of the existing fluorescence signal. DAPI-containing systems are suitable when both mounting and nuclear staining are intended to be completed in a single step, helping reduce workflow complexity and improve efficiency before observation.
(4) Limitations in use
For non-fluorescent chromogenic sections, these systems are generally unnecessary. If the experiment itself does not depend on fluorescence preservation, their advantages cannot be fully realized.
2.6 Neutral Balsam and Canada Balsam
(1) Composition and physical characteristics
Neutral balsam and Canada balsam are traditional resin-based mounting systems that generally require dehydration and clearing before use. After mounting, they form a transparent, hard, and relatively stable solid layer suitable for long-term storage and archiving.
(2) Applicable experimental types
These systems are best suited to HE staining, DAB-developed IHC, and routine paraffin sections. For specimens that require long-term preservation, repeated review, and pathological archive management, resin-based mounting media still offer clear advantages in stability.
(3) Application advantages
Their advantages lie in the firmness of the mounting layer, neat appearance, and excellent long-term storage performance, particularly for routine histological sections intended primarily for archiving and later review.
(4) Limitations in use
A prerequisite for their use is that the sample itself must tolerate dehydration and organic solvent exposure. Accordingly, resin-based systems are not suitable for fluorescent samples, lipid-stained specimens, or certain chromogenic systems sensitive to dehydration and clearing.
3 Selection Strategies in Different Experimental Scenarios
3.1 Immunofluorescence and Multiplex Fluorescence Experiments
For IF, FISH, and multiplex fluorescence experiments, the primary criterion in selecting a mounting system is whether it can protect fluorescence signals, rather than simple transparency or convenience of use. In fluorescence experiments, the main post-mounting risk is not coverslip displacement, but rapid signal loss caused by photobleaching.These experiments can be evaluated using the following logic:
(1) If independent nuclear staining has already been completed, a DAPI-free antifade mounting medium should be preferred.
(2) If mounting and nuclear staining are to be completed in one step, a DAPI-containing antifade mounting medium may be selected.
(3) If the sample requires transport, repeated observation, or storage for a certain period, a PVP-based antifade system may be preferred.
3.2 HE and Routine IHC Chromogenic Experiments
For HE staining, routine IHC, DAB-developed sections, and related assays, the evaluation focus is no longer fluorescence protection, but rather transparency, tissue boundary clarity, and long-term storage stability.In such cases, the following logic may be used:
(1) When a classical histological appearance and good transparency are desired, Kisser mounting medium may be preferred.
(2) When lower toxicity and suitability for batch processing are prioritized, PVP mounting medium may be considered.
(3) If standard dehydration and clearing have already been completed and long-term archiving is the goal, neutral balsam or Canada balsam may be used.
3.3 Frozen Sections and Lipid Staining
For Oil Red O staining, frozen sections, and certain enzyme histochemistry samples, the primary requirement in mounting is to avoid damage to sample components by organic solvents.Accordingly:
(1) For Oil Red O staining, water-based mounting systems such as glycerol gelatin should be preferred.
(2) For frozen sections that are not suitable for dehydration and clearing, water-soluble mounting media should also be prioritized.
(3) If frozen sections are to be examined by fluorescence microscopy, antifade mounting systems should still be preferred rather than ordinary glycerol gelatin.
Table 2. Recommended Mounting Choices for Different Experimental Scenarios
Experimental Scenario | Preferred Mounting System | Basis for Selection | Main Precautions |
Immunofluorescence (IF) | Antifade mounting medium | Protect fluorescence signals | Avoid light exposure throughout; do not use ordinary resin media or ordinary glycerol gelatin |
FISH | Antifade mounting medium | Protect nucleic acid probe fluorescence | Pay attention to sample drying rate and sealing after mounting |
Fluorescence experiments with nuclear staining | DAPI-containing antifade medium | Simplifies workflow | Avoid duplicating an existing nuclear staining strategy |
Routine HE staining | Kisser mounting medium or resin-based mounting medium | Transparency and archival stability | Choose according to whether dehydration and clearing have been completed |
Routine IHC (DAB development) | Kisser mounting medium or resin-based mounting medium | Stable chromogenic products and suitability for archiving | No need to pursue antifade performance |
Frozen sections | Water-soluble mounting medium | Avoid damage from organic solvents | Further subdivide according to whether fluorescence observation is involved |
Oil Red O staining | Glycerol gelatin | Preserves lipids | Xylene-based resin systems must not be used |
Fluorescent samples requiring repeated observation | PVP antifade mounting medium | Stable film formation and convenient storage | Protect from light and seal the edges |
4 Key Factors Affecting Mounting Quality
4.1 Surface Condition of the Sample
Before mounting, the sample should remain moist but free of obvious excess liquid. If too much PBS or other buffer remains on the sample surface, the mounting medium will be diluted and the uniformity of the mounting layer will be affected. If the sample surface becomes too dry, local refractive heterogeneity or bubbles are more likely to occur.
A more reliable approach is to keep the sample moist, absorb excess liquid from the edges and surface, and avoid allowing the sample to air-dry completely before mounting.
4.2 Coverslip Placement
Bubbles, local gaps, and incomplete edge coverage are often not caused by the mounting medium itself, but by improper coverslip placement. A better method is to lower the coverslip slowly from one side so that the mounting medium spreads naturally, rather than dropping it vertically onto the sample.
4.3 Refractive Matching and Image Quality
A mounting medium determines not only whether the sample can be sealed, but also whether the specimen can be clearly visualized. The degree of refractive matching among the mounting layer, slide, coverslip, and sample directly affects imaging clarity, background uniformity, and edge sharpness. This is particularly important in high-magnification observation and fluorescence localization experiments.
4.4 Curing Behavior and Storage Requirements
(1) Immediate-view systems are more suitable for rapid observation.
(2) Slowly curing systems are more suitable for short- to medium-term storage.
(3) Resin-based hard-sealing systems are more suitable for long-term archiving.
5 Products Related to Mounting System Research
5.1 Reagent Product Table for Mounting Systems
Product Category | Catalog No. | Name | Grade and Purity | Suitable Research Use/Application |
Glycerol-Based Routine Sealing Medium | Glycerol-Ethanol Mounting Medium | Suitable for Immunofluorescence(IF); BioReagent; for microscopy; Suitable for Immunohistochemistry(IHC) | Suitable for rapid mounting and routine observation in aqueous systems; applicable to IF/IHC samples that do not require strong long-term antifade protection | |
Routine Mounting Medium | mounting medium | Unscented | Suitable for routine slide mounting and general morphologic observation; can be used as a basic mounting medium | |
Routine Mounting Medium | Fischor Mounting Medium | BioReagent; for microscopy; Suitable for Immunofluorescence(IF); Suitable for Immunohistochemistry(IHC) | Suitable for routine IF/IHC sample mounting, balancing pre-imaging observation with short- to medium-term preservation | |
Glycerol-Based Routine Mounting Medium | Routine Glycerol Mounting Medium | BioReagent; for microscopy; Suitable for Immunofluorescence(IF); for fluorescence analysis | Suitable for short-term fluorescence observation and routine mounting under conditions that do not require strong antifade performance | |
Glycerol-Based Buffered Mounting Medium | Glycerol PBS Mounting Medium | BioReagent; for microscopy; Suitable for Immunofluorescence(IF); for fluorescence analysis | Suitable for maintaining samples in an aqueous environment; commonly used for immediate observation of cell smears and fluorescent tissue samples | |
Polyvinyl Alcohol-Based Mounting Medium | Polyvinyl Alcohol Glycerol Mounting Medium | BioReagent; for microscopy; Suitable for Immunofluorescence(IF); Suitable for Immunohistochemistry(IHC) | Suitable for routine IF/IHC samples requiring good film-forming properties and coverslip stability | |
Polyvinyl Alcohol Antifade Mounting Medium | Polyvinyl Alcohol Anti-Fluorescence Quenching Mounting Medium | Suitable for Immunofluorescence(IF); BioReagent; for microscopy; for fluorescence analysis | Suitable for fluorescent samples requiring repeated observation, imaging, or short- to medium-term storage, with both antifade protection and mounting stability | |
Natural Gum-Based Mounting Medium | Gum Arabic Glycerol Mounting Medium | BioReagent; for microscopy; Suitable for microbiology; Suitable for Immunofluorescence(IF); Suitable for Immunohistochemistry(IHC) | Suitable for routine slide mounting requiring a certain degree of adhesion and sealing stability | |
Antifade Mounting Medium | Antifluorescent quencher | — | Suitable for routine IF and fluorescence analysis, with emphasis on reducing photobleaching and extending the imaging window | |
Enhanced Antifade Mounting Medium | Enhanced Antifade Mounting Medium | BioReagent; for fluorescence analysis; Suitable for Immunofluorescence(IF) | Suitable for fluorescent samples requiring high exposure, multichannel imaging, or repeated observation, with enhanced signal-protection capability | |
Antifade Mounting Solution | Antifade Mounting Medium | — | Suitable for routine mounting of fluorescent sections, reducing fluorescence decay and improving observation stability | |
PI-Containing Antifade Mounting Solution | AntiFade Mounting Medium (with PI) | BioReagent; Suitable for Immunofluorescence(IF); Suitable for Immunohistochemistry(IHC) | Suitable for mounted observation requiring simultaneous nucleic acid/dead-cell-associated red fluorescence indication | |
Hoechst 33342-Containing Antifade Mounting Solution | AntiFade Mounting Medium (with Hoechst 33342) | BioReagent; Suitable for Immunohistochemistry(IHC); Suitable for Immunofluorescence(IF) | Suitable for fluorescence assays requiring one-step mounting and nuclear staining, facilitating nuclear localization and multichannel imaging | |
Hoechst 33342- and PI-Containing Antifade Mounting Solution | AntiFade Mounting Medium (with Hoechst 33342 and PI) | BioReagent; Suitable for Immunohistochemistry(IHC); Suitable for Immunofluorescence(IF) | Suitable for multiplex fluorescence assays requiring simultaneous observation of nuclear localization and PI signals | |
DAPI-Containing Antifade Mounting Solution | AntiFade Mounting Medium (with DAPI) | BioReagent; Suitable for Immunofluorescence(IF); Suitable for Immunohistochemistry(IHC) | Suitable for routine IF assays requiring one-step mounting and nuclear staining, facilitating rapid imaging | |
Routine Chromogenic Mounting Medium | Environmentally Friendly Mounting Medium | BioReagent; for microscopy; Suitable for Immunohistochemistry(IHC) | Suitable for routine chromogenic sections and IHC sample archiving, with emphasis on operational convenience and standard preservation | |
PVP Mounting Medium | Polyvinylpyrrolidone Mounting Medium | — | Suitable for routine tissue sections, cell smears, and teaching samples, balancing film-forming properties with relatively low volatility | |
Kisser-Type Mounting Medium | Kisser's Mounting Medium | BioReagent; ready-to-use; Suitable for Immunohistochemistry(IHC); Suitable for Immunofluorescence(IF) | Suitable for routine morphologic observation, non-intensive continuous exposure conditions, and general IHC mounting | |
Glycerol Gelatin Mounting Medium | Glycerol Jelly Mounting Medium | Suitable for Immunofluorescence(IF); BioReagent; ready-to-use; Suitable for Immunohistochemistry(IHC); sterile | Suitable for frozen sections, lipid-related staining, or samples requiring preservation in an aqueous environment | |
Kisser-Type Mounting Medium | Kisser's Mounting Medium(phenol free) | BioReagent; Suitable for Immunohistochemistry(IHC); Suitable for Immunofluorescence(IF) | Suitable for routine mounting applications where phenolic background interference is to be avoided, while supporting both IHC and general fluorescence observation |
5.2 Table of Chemical Components Related to Mounting Formulations and Functions
Product Category | Name | CAS No. | Corresponding Mounting System / Function | Research Direction / Use |
Base Medium | Glycerol | Water-soluble mounting matrix; improves refractive index | Used in glycerol-based, glycerol gelatin-based, and selected antifade mounting systems to maintain sample hydration and optical transparency | |
Gel Matrix | Gelatin | Thermosensitive gel scaffold | Used in glycerol gelatin and Kisser-type systems to enhance sealing performance and tissue adhesion stability | |
Film-Forming Polymer | Polyvinylpyrrolidone (PVP) | Film formation and protective layer construction | Used in PVP mounting media and PVP-based antifade systems to improve coverslip stability and sample preservation | |
Film-Forming Polymer | Polyvinyl alcohol (PVA) | Film formation and auxiliary matrix for anti-photobleaching systems | Commonly used in antifade mounting systems to improve film uniformity and long-term observation stability | |
Natural Gum Matrix | Gum arabic | Natural viscosity-enhancing and sealing matrix | Used in natural gum-based mounting systems to improve section sealing and edge stability | |
Antifade Reagent | 1,4-Diazabicyclo[2.2.2]octane (DABCO) | Reactive oxygen species scavenging; reduces photobleaching | Suitable for fluorescence assays such as IF and FISH; a classical additive for antifade mounting formulations | |
Antifade Reagent | p-Phenylenediamine | Free radical scavenging; fluorescence protection | Used to reduce fluorescence signal decay rate; commonly found in early antifade system studies | |
Antifade Reagent | n-Propyl gallate | Antioxidation; delays fluorescence quenching | Suitable for optimization of fluorescent mounting formulations; commonly used to improve signal retention under illumination | |
Nuclear Dye | DAPI | DNA nuclear staining | Used in DAPI-containing antifade mounting media to achieve simultaneous mounting and nuclear staining | |
Nuclear Dye | Hoechst 33342 | DNA nuclear staining | Used in Hoechst-containing mounting media; widely applied for nuclear localization in live or fixed cells | |
Nucleic Acid Dye | Propidium iodide (PI) | Nucleic acid staining / dead-cell-associated indicator | Used in PI-containing mounting media or combined nuclear-staining mounting formulations; suitable for cell-state interpretation and multichannel fluorescence imaging | |
Preservative / Background-Related Component | Phenol | Component associated with traditional Kisser-type systems | Associated with selected traditional mounting formulations; affects preservation, refractive behavior, and background characteristics | |
Resinous Mounting Component | Canada balsam | Resinous matrix for long-term sealing | Suitable for long-term preservation of H&E- and DAB-stained sections; not suitable for fluorescence experiments | |
Clearing-Related Solvent | Xylene | Clearing step before resin-based mounting | Suitable for resin-based mounting workflows, but damages fluorescence and lipid-containing samples; not appropriate for IF or Oil Red O-type experiments |
6 Common Problems and Control Points in Experimental Operation
6.1 Bubble Control
Bubbles not only interfere with observation, but may also lead to uneven local coverage of the sample, thereby affecting imaging and storage quality. The key to reducing bubbles lies in applying an appropriate volume of mounting medium and lowering the coverslip slowly. When necessary, bubbles can be gently guided toward the edge with the tip of forceps after coverslip placement.
6.2 Light Protection and Low-Temperature Storage of Fluorescent Samples
Even when antifade mounting medium is used, fluorescence may still decline significantly if mounted samples remain exposed to strong light or room temperature for prolonged periods. A more reliable approach is to protect samples from light throughout the workflow, image them as soon as possible after mounting, or store them at low temperature. If necessary, the edges of the coverslip should also be sealed.
6.3 Edge Cleaning after Resin-Based Mounting
Overflow of resin-based mounting medium can affect slide neatness and archival quality. Edge cleaning is best performed after the mounting medium has partially stabilized. A small amount of compatible solvent may be used to remove excess material, but contact with the sealing layer beneath the coverslip should be avoided.
6.4 Risks of Using Ordinary Glycerol Systems in Fluorescence Experiments
Ordinary glycerol or ordinary glycerol gelatin is not equivalent to an antifade system. Even if some fluorescence remains visible for a short time, such systems usually cannot support stable observation and are even less suitable for repeated imaging or delayed photography. This is one of the most commonly misjudged points in practical operation.
The essence of mounting medium selection lies in matching sample properties, imaging modality, and preservation objectives. Fluorescence experiments should prioritize antifade performance; lipid-containing and frozen samples should prioritize aqueous compatibility; routine chromogenic sections should place greater emphasis on transparency and long-term archival stability. Only by evaluating the mounting system within the context of the complete experimental workflow can one truly avoid losing value at the final mounting stage after successful upstream staining.
For more related articles, please see below:
[1] Analysis and handling of IHC common problems
[2] Immunohistochemistry (fluorescence) assay