The exploration of biological tissues amongst the kingdoms of nature spans centuries. Researchers have developed various methods of investigation to understand the composition, structure, and characteristics of organs. Preservation of organic matter plays an essential role in such studies.
Scientists have long stored tissues to inspect and prove their concepts as part of their long-standing routine. Conserving methods have contributed to most of what we know from molecule to whole-organism studies. The chemical and physical approaches led to the development of formaldehyde-based fixation and set it as the gold standard throughout the last century. As an alternative, fresh frozen tissues are also used since non-fixed samples are now frequently required for investigation (Sadler et al., 2009).
Rapid freezing helps to save time and does not require a lengthy fixation compared with formaldehyde (FA) incubation (Shi et al., 2008). Cold temperatures allow the preservation of DNA and RNA that can be further investigated (Hahn et al., 2023, Cui et al., 2024). Along with nucleic acids, freezing prevents the degradation of other molecules. Therefore, metabolomic, proteomic, sequencing and other studies can all be conducted using one piece of tissue (Vekaria et al., 2024).
Immunohistochemistry requires tissue fixation, but how can the best fixative be chosen? Used and well-liked by many, formalin-fixed tissues continue to bring robust results. However, new scientific progress requires a more flexible fixation to preserve other molecules that formaldehyde-based approaches do to a lesser extent.
Investigators worldwide ask us about the products’ performance in staining acetone or methanol fixed tissues. In this company, we strive to provide the best products for our customers, so we embarked on the journey of testing our products in conditions other than our standard fixation protocol with 4% formaldehyde (FA) for 24h. In this Featured Topic, we investigate the applicability of four fixatives: acetone, methanol, acetone-methanol (1:1) and 4% FA in fresh frozen tissue sections.
Despite the popularity of formaldehyde-based fixatives, some cellular regions are difficult to fix well for the downstream immunohistochemical investigation. High protein packing and dynamics in post-synaptic density (PSD) and axonal initial segment (AIS) are challenging to visualize even with high-resolution techniques (Eilts et al., 2023, Sun et al., 2021). Above 50% of PSD95 protein assemblies change within 1 hour in PSD (Morris et al., 2024). This, in turn, requires quick fixation that preserves tissue morphology and protein localization well. A high degree of protein crosslinking in this densely packed compartment frequently renders antibody binding epitopes inaccessible. Aware of these issues, we were interested in how different fixatives impact the result of immunostainings obtained with our products.
Located in the PSD, Shank proteins provide a scaffold for the structure and allow molecular trafficking that contributes to high molecular dynamics (Vyas et al., 2021). This leads to challenges in visualization. To explore whether fixatives improve signal, we used exemplary Shank1, Shank2, and Shank3 antibodies (and others, data not shown) from our product portfolio. While all Shank proteins share common domains, the expression patterns vary in the nervous system (Figure 1) (Ha et al., 2016, Woelfle et al., 2023). Alternative splicing and promoter sites make the length and composition of each more complex and allow a greater variety of Shanks. This leads to separate functions of Shank1, Shank2, and Shank3 isoforms.
Based on our experience, almost no Shank antibody gave good results with our standard fixation protocol, 4% formaldehyde for 24h. Therefore, we used fresh frozen tissue sections and applied various fixatives to determine the best protocol and to investigate how specific and adaptable the antibodies are.
Figure 1: Visualization of various Shank1, Shank2, and Shank3 isoforms based on their exon composition, domains and promoters. The Ankyrin Repeat Domain (ANK) binds to actin cytoskeleton proteins, contributing to structural stability. Ion channel regulation is possible through the SH3 domains, and together with the PDZ domain, they support scaffold binding and receptor coupling. A proline-rich region (PRO) connects to metabotropic glutamate receptors (calcium signaling) and contractin (synapse cytoskeletal remodeling). Critical for Shank oligomerization and cooperation with other domains is the Sterile Alpha Motif (SAM) (Figure adapted from Wan L & Liu D, et al., 2021).
The detailed step-by-step protocol on tissue preparation and protocol for fixation and immunostaining are available on the website.
Figure 2: Outlook of the experimental procedure. Shortly, the brains were extracted, snap-frozen and cut using a cryostat Leica CM3050 S. Next, the tissues were fixed with 4% FA, acetone, methanol, or a 1:1 acetone-methanol mixture. After that, the slices were stained with Shank antibodies and analyzed using Zeiss Axio Observer Z1 microscope.
Briefly, the animals were transcardially perfused with 30-50 ml cold 0.9% saline containing 17 U/mL heparin at a rate of 5 ml/min until the tissue was cleared of blood. Next, the samples were dissected and immersed in Tissue-Tek®, after which they were immediately snap-frozen in liquid nitrogen pre-cooled isopentane (Figure 2). Until the dissection, the tissues were stored at -80°C. Using a cryostat Leica CM3050 S the samples were cut 12 µm thick at -18-20°C and mounted on Superfrost™ Plus Microscope slides (Thermo Fischer Scientific). Eventually, the sections were dried for 3-5 min at room temperature (RT) and stored at -80°C.
On the day of the staining, the sections were taken from -80°C and rapidly warmed up for a few minutes at room temperature. After that, the tissue was processed as follows:
Afterward, the slides were washed three times for 10 min with Tris-buffered saline (TBS) at RT. Before or after fixation, the tissue was enclosed with a hydrophobic pen. The slides were then incubated with a blocking buffer solution (10% normal goat serum, 0.3% Triton X-100 in TBS). Next, the solution was removed and the sections were incubated with primary antibodies (1:500 in 5% normal goat serum, 0.3% Triton X-100 in TBS) overnight at +4°C in a wet chamber.
| Protein | Cat. no. | Species | Clonality | Format |
| Shank1 | 162 013 | Rabbit | Polyclonal | Affinity purified |
| 162 106 | Chicken | Polyclonal | Affinity purified | |
| 162 121 | Mouse | Monoclonal | Purified IgG | |
| 162 123 | Rabbit | Polyclonal | Affinity purified | |
| Shank2 | 162 202 | Rabbit | Polyclonal | Antiserum |
| 162 211 | Mouse | Monoclonal | Purified IgG | |
| Shank3 | 162 306 | Chicken | Polyclonal | Affinity purified |
| 162 311 | Mouse | Monoclonal | Purified IgG | |
| Shank1/2/3 | 162 105 | Guinea pig | Polyclonal | Affinity purified |
| 162 111 | Mouse | Monoclonal | Purified IgG |
Table 1. List of Shank antibodies used for investigation.
The next day, the slides were washed thrice for 10 min with TBS at RT. Next, the secondary antibody solution was added for one hour at RT in a dark place. The following secondary antibodies were used: 706-165-148, 115-165-146, 111-165-144, 703-165-155 (Jackson ImmunoResearch). The slides were then washed thrice for 10 min with TBS at RT and incubated with DAPI solution (1:20,000 in PBS) to stain nuclei. Finally, the slides were washed once with tap water, the hydrophobic pen markings were removed, and the slides were mounted using Aqua-Poly/Mount (Polysciences, Inc.). The following day, the staining was examined at the Zeiss Axio Observer Z1 microscope.
Equal exposure times were used to produce the images to assess the influence of fixatives on signal intensity. The company researcher chose staining with the strongest signal among various fixatives and set the exposure time later used for visualizing other slides. Pictures of the hippocampus and cerebellum were taken with 20x objective and used for further analysis. After that, the pictures were uniformly edited to ensure the visibility and comparison of the images. A few slides that produced very weak signals were edited individually to reveal the immunosignal in the figures. This is mentioned in the figure legend. The markings “+++”, “++” and “+” were used to assess based on the subjective observation of the signal intensity. The main focus of the work was to determine which fixatives yield specific antibody signals while minimizing non-specific background staining.
Fixation with acetone, methanol, acetone-methanol (1:1) mixture, and FA led to various signal intensities of Shank1, Shank2 and Shank3 proteins stained with available antibodies (see Table 1).
Shank1 immunostaining with acetone fixation showed the best signal intensity (Figure 3). The 2nd strongest signal was detected in FA-fixed tissues. Shank1 antibodies cat. no. 162 013 and cat. no. 162 123 showed a good signal in methanol-fixed tissues, but not cat. no. 162 106 and cat. no. 162 121. All conditions showed a varying signal intensity; however, acetone-methanol and methanol fixation in separate cases resulted in nuclear staining (N). Additionally, immunostaining with the cat. no. 162 121 antibody showed non-specific blood vessel staining (BV), possibly caused by the secondary anti-mouse antibodies reacting with endogenous mouse IgGs remaining in residual blood not fully cleared during perfusion.
Figure 3: Immunostaining results with Shank1 antibodies. Microscopy images illustrate the effects of acetone, acetone-methanol (1:1), methanol, and 4% FA fixation on indirect immunostaining of fresh frozen mouse hippocampus sections with different Shank1 antibodies (1:500, greyscale). “+++” – very good, “++” – good, “+” – satisfactory staining, “N” – non-specific staining of nuclei, “BV” – non-specific staining of blood vessels. Exposure times: cat. no. 162 013 – 170 ms; cat. no. 162 123 – 62 ms; cat. no. 162 106 – 143 ms; cat. no. 162 121 – 67 ms. All pictures underwent the same editing process, with uniform parameters per antibody, except cat. no. 162 121 postfixed with methanol (signal intensity was increased for visualization purposes). Scale bar: 100 µm.
Immunostaining with Shank2 (Figure 4) and Shank3 (Figure 5) antibodies showed a similar trend to Shank1 staining. Acetone-methanol and methanol-fixed fixation resulted in non-specific staining of blood vessels and/or nuclei, while acetone and FA fixation led to the strongest signals. Pan-Shank 1/2/3 antibodies showed a strong specific signal in the mouse brain in all conditions, highest with acetone fixation (Figure 6).
Figure 4: Immunostaining results with Shank2 antibodies. Microscopy images illustrate the effects of acetone, acetone-methanol (1:1), methanol, and 4% FA fixation on indirect immunostaining of fresh frozen mouse hippocampus sections with Shank2 antibodies (1:500, greyscale). “+++” – very good, “++” – good, “+” – satisfactory staining, “N” – non-specific staining of nuclei. Exposure times: cat. no. 162 211 - 447 ms; cat. no. 162 202 – 91 ms. All pictures underwent the same editing process, with uniform parameters per antibody. Scale bar: 100 µm.
Figure 5: Immunostaining results with Shank3 antibodies. Microscopy images illustrate the effects of acetone, acetone-methanol (1:1), methanol, and 4% FA fixation on indirect immunostaining of fresh frozen mouse hippocampus sections with Shank3 antibodies (1:500, greyscale). “+++” – very good, “++” – good, “+” – satisfactory staining, “N” – non-specific staining of nuclei, “BV” – non-specific staining of blood vessels. Exposure times: cat. no. 162 306 - 128 ms; cat. no. 162 311 – 319 ms. All pictures underwent the same editing process, with uniform parameters per antibody. Scale bar: 100 µm.
Figure 6: Immunostaining results with Pan-Shank1/2/3 antibodies. Microscopy images illustrate the effects of acetone, acetone-methanol (1:1), methanol, and 4% FA fixation on indirect immunostaining of fresh frozen mouse hippocampus sections with antibodies targeting Shank1, Shank2 and Shank3 proteins jointly (cat. no. 162 111, cat. no. 162 105; 1:500). “+++” – very good, “++” – good, “+” – satisfactory staining. Exposure times: cat. no. 162 111 - 115 ms; cat. no. 162 105 – 156 ms. All pictures underwent the same editing process, with uniform parameters per antibody, except cat. no. 162 105 postfixed with acetone-methanol (signal intensity increased for visualization purposes). Scale bar: 100 µm.
Figure 7 shows the antibody specificity in the mouse cerebellum. While Shank1 and Shank2 are more prominent in the molecular layer, Shank3 is on the other hand located mostly in the granular layer. Antibodies cat. no. 162 123, cat. no. 162 106, cat. no. 162 202, and cat. no. 162 306 show varying levels of cross-reactivity with other Shank proteins, which is indicated in the remarks sections on the web page.
Figure 7: Specificity and cross-reactivity of Shank antibodies. Microscopy images showing the comparison of Shank1, Shank2 and Shank3 antibodies’ specificity and cross-reactivity using indirect immunostaining of fresh frozen mouse cerebellum sections post-fixed with acetone. All pictures underwent the same editing process, with uniform parameters per antibody. Scale bar: 100 µm.
Our standard fixation used for the validation of newly developed antibodies is perfusion followed by a 24h post-fixation with 4% formaldehyde (FA). We appreciate the good tissue preservation and handling of formaldehyde-fixed tissue. Many antibodies of our product range show very good staining results after 24h formaldehyde fixation. In some cases, the epitopes are masked by FA crosslinking, and antigen retrieval is needed to open the epitope for antibody binding. But some antibodies directed against special cellular regions like PSD or AIS tend to perform poorly after long formaldehyde fixation. Therefore, we started with fresh frozen sections for two reasons. First, to identify a better fixation method for antibodies targeting densely packed and dynamic structures, and second, to provide customers with data on antibody performance following methanol or acetone fixation, facilitating co-studies of proteins and RNA or DNA.
Throughout the entire series of experiments, acetone consistently yielded the strongest signal intensity compared to other fixatives. The second most successful fixative was a brief 15 min fixation with FA. It allowed detection and signal intensity superior to the 24 hour standard fixation protocol. However, when the antibody produces a weak signal in FA-fixed tissue, one has to consider a possible detection of FA-induced non-specific autofluorescent background signal from fiber tracts. On average, methanol and methanol-acetone mixture produced the weakest specific staining and additional non-specific signals of nuclei or blood vessels. However, these fixatives can still be employed in tissue staining using 162 123, 162 111, 162 105 antibodies. It is still unclear how exactly methanol-containing fixatives lead to undesirable nuclear staining in some cases and why not in others.
Why not always using fresh frozen sections? It is hard to reach high tissue preservation and integrity with fresh frozen sections. This is why scientists have to choose the fixation method depending on their experiments. While methanol is commonly used for DNA and RNA analyses, acetone is used less frequently, and despite progress in using special methodology, FA-fixed tissues are still unfavorable (Hahn et al., 2023, Cui et al., 2024).
We try to support researchers by providing information on antibody performance under different fixation methods. On our webpage, IHC is defined as immunohistochemistry based on our standard protocol using tissue fixed with formaldehyde for 24 hours, sectioned with a cryostat or vibratome. We indicate whether subsequent antigen retrieval (AGR) is recommended. Furthermore, we have implemented IHC-Fr (IHC on fresh frozen sections), and we are consistently expanding the available data on tested antibodies. Our application filters, like the one for IHC-Fr, enable easy identification of antibodies validated for use on snap-frozen sections.
| Cat. No. | Product Description | Application | Quantity | Price | Cart |
|---|
| 162 013 | Shank1, rabbit, polyclonal, affinity purifiedaffinity K.O. | WB ICC IHC-Fr | 50 µg | $460.00 |   |
| 162 106 | Shank1, chicken, polyclonal, affinity purifiedaffinity | WB ICC IHC-Fr ExM | 50 µg | $385.00 |  |
| 162 121 | Shank1, mouse, monoclonal, purified IgG IgG | ICC IHC-P IHC-Fr | 100 µg | $420.00 | |
| 162 123 | Shank1, rabbit, polyclonal, affinity purifiedaffinity | WB ICC IHC-Fr | 50 µg | $380.00 | |
| 162 105 | Shank1/2/3, Guinea pig, polyclonal, affinity purifiedaffinity | ICC IHC-Fr | 50 µg | $465.00 | |
| 162 111 | Shank1/2/3, mouse, monoclonal, purified IgG IgG | ICC IHC IHC-P IHC-Fr DNA-PAINT | 100 µg | $420.00 | |
| 162 202 | Shank2, rabbit, polyclonal, antiserumantiserum K.O. K.D. | WB ICC IHC IHC-Fr | 200 µl | $360.00 | |
| 162 211 | Shank2, mouse, monoclonal, purified IgG IgG | ICC IHC IHC-Fr | 100 µg | $420.00 | |
| 162 306 | Shank3, chicken, polyclonal, affinity purifiedaffinity | WB ICC IHC-Fr | 50 µg | $385.00 | |
| 162 311 | Shank3, mouse, monoclonal, purified IgG IgG | WB ICC IHC-Fr | 100 µg | $420.00 | |
Author: Dr. Yuliia Tereshchenko
Co-author: Dr. Liane Wüstefeld
Scientists at the Histology Department
Yuliia is part of the Histology Department and has extensive experience in immunostaining of various organ systems. In SYSY Antibodies, she develops and optimizes specific staining procedures for protocols using alternative fixatives for fresh frozen and perfused tissues. As a part of her daily tasks, she also validates in-house developed antibodies and processes tissues for downstream analyses.
Akane et al., 2013: Methacarn as a whole brain fixative for gene and protein expression analyses of specific brain regions in rats. PMID: 23665942
Cui et al., 2024: Large field of view and spatial region of interest transcriptomics in fixed tissue. PMID: 39164496
Eilts et al., 2023: Enhanced synaptic protein visualization by multicolor super-resolution expansion microscopy. PMID: 37886043
Evers et al., 2011: The effect of formaldehyde fixation on RNA: optimization of formaldehyde adduct removal. PMID: 21497290
Galli et al., 2014: Effects of tissue fixation on coherent anti-Stokes Raman scattering images of brain. PMID: 24365991
Ha et al., 2016: Cerebellar Shank2 Regulates Excitatory Synapse Density, Motor Coordination, and Specific Repetitive and Anxiety-Like Behaviors. PMID: 27903723
Hahn et al., 2023: Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality. PMID: 37274189
Hoetelmans et al., 2001: Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy. PMID: 11759062
Kaku et al., 1983: Comparison of formalin- and acetone-fixation for immunohistochemical detection of carcinoembryonic antigen (CEA) and keratin. PMID: 6195915
Morris et al., 2024: Sequential replacement of PSD95 subunits in postsynaptic supercomplexes is slowest in the cortex. PMID: 39570289
Rahman et al., 2022: Alcoholic fixation over formalin fixation: A new, safer option for morphologic and molecular analysis of tissues. PMID: 35002406
Sadler et al., 2009: Snap-frozen brain tissue sections stored with desiccant at ambient laboratory conditions without chemical fixation are resistant to degradation for a minimum of 6 months. PMID: 19521279
Scandella et al., 2020: A novel protocol to detect green fluorescent protein in unfixed, snap-frozen tissue. PMID: 32887893
Schmidt et al., 2003: Paraformaldehyde fixation induces a systematic activation of platelets. PMID: 12944245
Shi et al., 2008: Evaluation of the value of frozen tissue section used as "gold standard" for immunohistochemistry. PMID: 18285257
Sun et al., 2021: The prevalence and specificity of local protein synthesis during neuronal synaptic plasticity. PMID: 34533986
Vekaria et al., 2024: An efficient and high-throughput method for the evaluation of mitochondrial dysfunction in frozen brain samples after traumatic brain injury. PMID: 38983247
Vyas et al., 2021: Shankopathies in the Developing Brain in Autism Spectrum Disorders. PMID: 35002604
Wan & Liu, et al, 2021: Association of SHANK Family with Neuropsychiatric Disorders: An Update on Genetic and Animal Model Discoveries. PMID: 33595806
Woelfle et al., 2023: Expression profiles of the autism-related SHANK proteins in the human brain. PMID: 37953224
Yan et al., 2010: The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods. PMID: 20888418
