Immunostaining Protocols | Immunodetection | Protein | Biochemistry

Time-Lapse Into Immunofluorescence Imaging Using a Gridded Dish

NL Nick Lang

CC Catherine G. Chu

AS Andrew D. Stephens

269 Views

Feb 20, 2026

Time-lapse into immunofluorescence (TL into IF) imaging combines the wealth of information acquired during live-cell imaging with ease of access for static immunofluorescence markers. In the field of mechanobiology, connecting live and static imaging to visualize cell biology dynamics is often troublesome. For instance, nuclear blebs are deformations of the nucleus that often rupture spontaneously, leading to changes in the molecular composition of the nucleus and the nuclear bleb. Current techniques to connect cellular dynamics and their downstream effects via live-cell imaging, followed by immunofluorescence, often require third-party analysis programs or stage position measurements to accurately track cells. This protocol simplifies the connection between live and static imaging by utilizing a gridded imaging dish. In our protocol, cells are plated on a dish with an engraved coordinate plane. Individual cells are then matched from when the time-lapse ends to the immunofluorescence images simply by their known coordinate location. Overall, TL into IF offers a straightforward method for connecting dynamic live-cell with static immunofluorescence imaging, in an easy and accessible tool for cell biologists.

Immunofluorescence for Detection of TOR Kinase Activity In Situ in Photosynthetic Organisms

AL Ana P. Lando

MD María A. De Marco

AC Andrea C. Cumino

GM Giselle M. A. Martínez-Noël

0 Q&A

1901 Views

Dec 20, 2024

The target of rapamycin (TOR) is a central hub kinase that promotes growth and development in all eukaryote cells. TOR induces protein synthesis through the phosphorylation of the S6 kinase (S6K), which, in turn, phosphorylates ribosomal S6 protein (RPS6) increasing this anabolic process. Therefore, S6K and RPS6 phosphorylation are generally used as readouts of TOR activity. Protein phosphorylation levels are measured by a western blot (WB) technique using an antibody against one specific phosphosite in cell extracts. However, at the tissue/cell-specific level, there is a huge gap in plants due to the lack of alternative techniques for the evaluation of TOR activity as there are for other organisms such as mammals. Here, we describe an in vivo protocol to detect S6K phosphorylation in tissues/cells of model photosynthetic organisms such as Arabidopsis thaliana and Chlamydomonas reinhardtii. Our proposed method consists of the immunolocalization of a phosphorylated target of TOR kinase using a fluorescent secondary antibody by confocal microscopy. The protocol involves four main steps: tissue/cell fixation, permeabilization, and incubation with primary and secondary antibodies. It is an easy technique that allows handling different samples at the same time. In addition, different ultrastructural cell markers can also be used, such as for nucleus and cell wall detection, allowing a detailed analysis of cell morphology. To our knowledge, this is the first protocol to detect TOR activity in situ in photosynthetic organisms; we consider that it will pave the research on the TOR kinase, opening new possibilities to better understand its complex signaling.

Non-separate Mouse Sclerochoroid/RPE/Retina Staining and Whole Mount for the Integral Observation of Subretinal Layer

SL Sung-Jin Lee

Soo-Young Kim

0 Q&A

4549 Views

Jan 5, 2021

The subretinal layer between retinal pigment epithelium (RPE) and photoreceptors is a region involved in inflammation and angiogenesis during the procession of diseases such as age-related macular degeneration. The current protocols of whole mounts (retina and RPE) are unable to address the intact view of the subretinal layer because the separation between retina and RPE is required, and each separate tissue is then stained. Non-separate Sclerochoroid/RPE/Retina whole mount staining was recently developed and reported. The method can be further combined and optimized with melanin bleaching and tissue clearing. Here, we describe steps of both non-pigmented and pigmented mouse Sclerochoroid/RPE/Retina whole mount including eyeball preparation, staining, mounting and confocal scanning. In addition, we present the confocal images of RPE, subretinal microglia and the neighboring photoreceptors in Sclerochoroid/RPE/Retina whole mounts.

Quantification of Cutaneous Ionocytes in Small Aquatic Organisms

GK Garfield T. Kwan

SF Shane H. Finnerty

NW Nicholas C. Wegner

Martin Tresguerres

0 Q&A

5936 Views

May 5, 2019

Aquatic organisms have specialized cells called ionocytes that regulate the ionic composition, osmolarity, and acid/base status of internal fluids. In small aquatic organisms such as fishes in their early life stages, ionocytes are typically found on the cutaneous surface and their abundance can change to help cope with various metabolic and environmental factors. Ionocytes profusely express ATPase enzymes, most notably Na⁺/K⁺ ATPase, which can be identified by immunohistochemistry. However, quantification of cutaneous ionocytes is not trivial due to the limited camera’s focal plane and the microscope’s field-of-view. This protocol describes a technique to consistently and reliably identify, image, and measure the relative surface area covered by cutaneous ionocytes through software-mediated focus-stacking and photo-stitching–thereby allowing the quantification of cutaneous ionocyte area as a proxy for ion transporting capacity across the skin. Because ionocytes are essential for regulating ionic composition, osmolarity, and acid/base status of internal fluids, this technique is useful for studying physiological mechanisms used by fish larvae and other small aquatic organisms during development and in response to environmental stress.

Biofilm Assays on Fibrinogen-coated Silicone Catheters and 96-well Polystyrene Plates

Cristina Colomer-Winter

José A. Lemos

Ana L. Flores-Mireles

0 Q&A

7685 Views

Mar 20, 2019

Biofilm formation is a well-known bacterial strategy that protects cells from hostile environments. During infection, bacteria found in a biofilm community are less sensitive to antibiotics and to the immune response, often allowing them to colonize and persist in the host niche. Not surprisingly, biofilm formation on medical devices, such as urinary catheters, is a major problem in hospital settings. To be able to eliminate such biofilms, it is important to understand the key bacterial factors that contribute to their formation. A common practice in the lab setting is to study biofilms grown in laboratory media. However, these media do not fully reflect the host environment conditions, potentially masking relevant biological determinants. This is the case during urinary catheterization, where a key element for Enterococcus faecalis and Staphylococcus aureus colonization and biofilm formation is the release of fibrinogen (Fg) into the bladder and its deposition on the urinary catheter. To recapitulate bladder conditions during catheter-associated urinary tract infection (CAUTI), we have developed a fibrinogen-coated catheter and 96-well plate biofilm assay in urine. Notably, enterococcal biofilm factors identified in these in vitro assays proved to be important for biofilm formation in vivo in a mouse model of CAUTI. Thus, the method described herein can be used to uncover biofilm-promoting factors that are uniquely relevant in the host environment, and that can be exploited to develop new antibacterial therapies.

Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells

FU Florent Ubelmann

Tatiana Burrinha

Claudia Guimas Almeida

0 Q&A

8168 Views

Dec 5, 2017

The established primary trigger of Alzheimer’s disease’s is β-amyloid (Aβ) (Mucke and Selkoe, 2012). Amyloid precursor protein (APP) endocytosis is required for Aβ generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes also depends on its recycling back to the plasma membrane (Koo et al., 1996; Ubelmann et al., 2017). The following recycling assay has been optimized to assess APP recycling by live murine Neuro2a cells, a neuroblastoma cell line (Ubelmann et al., 2017).

Indirect Immunofluorescence Assay in Chlamydomonas reinhardtii

Takashi Yamano

HF Hideya Fukuzawa

0 Q&A

13354 Views

Jul 5, 2016

Determining the protein localization is essential to elucidate its in vivo function. Fluorescence-tagged proteins are widely used for it, but it is sometimes difficult to express tagged proteins in Chlamydomonas. Alternatively, indirect immunofluorescence assay is also one of the widely used methods and many reports determining the localization of Chlamydomonas proteins using this method are published. Here, we introduce a protocol of indirect immunofluorescence assay adapted from our papers reporting LCIB (CO₂-recycling factor in the vicinity of pyrenoid; Yamano et al., 2010), LCI1 (plasma membrane-localized inorganic carbon transporter; Ohnishi et al., 2010), HLA3 (plasma membrane-localized ABC-type bicarbonate transporter; Yamano et al., 2015), and LCIA (chloroplast envelope anion channel; Yamano et al., 2015) in Chlamydomonas reinhardtii. The protocol described here could be useful for observing the protein of interest in other algae cells.

Immunolocalization of Proteins in Corals: the V-type H⁺-ATPase Proton Pump

KB Katie L. Barott

Martin Tresguerres

0 Q&A

11113 Views

Sep 5, 2015

Here we describe the immunolocalization of a membrane-bound proton pump, the V-type H⁺-ATPase (VHA), in tissues and isolated cells of scleractinian corals. Immunolocalization of coral proteins requires additional steps not required for various model organisms, such as decalcification of the coral skeleton for immunohistochemistry or removal of cells away from the skeleton for immunocytochemistry. The tissue and cell preparation techniques described here can be adapted for localization of other coral proteins, provided the appropriate validation steps have been taken for the primary antibodies and species of coral used. These techniques are important for improving our understanding of coral cell physiology

Combined in situ Hybridization/Immunohistochemistry (ISH/IH) on Free-floating Vibratome Tissue Sections

Manuel E. Lopez

0 Q&A

15559 Views

Sep 20, 2014

In situ hybridization and immunostaining are common techniques for localizing gene expression, the mRNA and protein respectively, within tissues. Both techniques can be applied to tissue sections to achieve similar goals, but in some cases, it is necessary to use them together. For example, complement C1q is a secreted protein complex that can target the innate immune response during inflammation. Complement has been found to be elevated early and before severe neurodegeneration in several disease models. Thus, complement may serve as an important marker for disease progression and may contribute to the pathology under certain conditions. Since complement is a secreted complex, immunostaining for C1q does not necessarily reveal where compliment is produced. In situ hybridization for complement components, C1q a, b, or c mRNA, is ideal to mark complement producing cells in tissue. In situ hybridization can be coupled with cell-type-specific immunostaining for accurate identification of the cell types involved. Protein localization and mRNA localization together can reveal details as to the relationship between complement producing and complement target cells within disease tissues. Here we outline the steps for combined in situ hybridization and immunostaining on the same tissue section. The protocol outlined here has been designed for detection of complement C1q in neurons and microglia in the mouse brain.

Provided here are two approaches for combined ISH/IH. In the 1st example, in situ hybridization of C1q mRNA is performed together with fluorescent detection of Purkinje neuron cell bodies using Calbindin-D28K antibody. In the 2nd example, C1q mRNA in situ is performed together with 3,3’-diaminobenzidine (DAB) detection of microglia using CD68 antibody. Please note that modifications to the protocol may be needed for the use of distinct probes and antibodies, as well as alternate tissue-processing methods that are not specified herein. For appropriate examples of procedure results, please see images published in Lopez et al.. (2012).

Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites

AH Arlett Heiber

SR Silke Retzlaff

TS Tobias Spielmann

0 Q&A

9967 Views

Jun 5, 2014

This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined using standard transmission electron microscopy (EM). Pre-embedding EM is a common method where the antibodies are introduced before embedding and sectioning. This method avoids the problem that antigens are often difficult to detect on EM-sections after embedding. In the method described here antigens in the parasite-infected host cell are detected. Entry of the antibody is made possible through permeabilisation of the host cell with tetanolysin. In principle this method could also be used to detect antigens within the parasite if the sample is appropriately fixed and permeabilised before addition of the relevant antibody. While access of the antibody will avoid the detection problems often seen with post-embedding methods, this procedure will produce comparably poorer morphology.